Novel pharmaceutical composition containing hydroxamic acid derivative or salt thereof

ABSTRACT

This pharmaceutical composition contains a hvdroxamic acid derivative, or a salt thereof, and a solubilizer, said hydroxamic acid derivative being selected from among (2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-hydroxy-N′,2- dimethylmalonamide, (2S)-2-((4((4-((1R)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-hydroxy-N′,2-dimethylmalonamide, and (2S)-N-hydroxy-2-((4-((4-((1S)-1-hydroxy-2-methoxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N′,2-dimethylmalonamide. The pharmaceutical composition demonstrates strong antibacterial activity, has excellent solubility in water, and is useful as a drug.

TECHNICAL FIELD

The present invention relates to pharmaceutical compositions comprisingnovel hydroxamic acid derivatives or the salt thereof.

BACKGROUND ART

Gram-negative bacteria have an outer membrane composed of a lipidbilayer, which does not exist in Gram-positive bacteria, and thereforetend to have stronger drug resistance, as compared to Gram-positivebacteria. Gram-negative bacteria are also known to have a plurality ofdrug efflux proteins, which are involved in drug resistance(Antimicrobial Resistance, 2002, Mar. 1, 34, pp. 634-640).

Among Gram-negative bacteria, Pseudomonas aeruginosa, in particular, hasa strong tendency to show intrinsic resistance to various antimicrobialdrugs. In recent years, Psendomonas aeruginosa which has gainedresistance to carbapenem drugs, quinolone drugs, aminoglycoside drugs,or the like has been often isolated in medical settings (J. Antimicrob.Chemother., 2003, Vol. 51, pp. 347-352). Moreover, multi-drug resistantPseudomonas aeruginosa has been isolated (Jpn. J. Antibiotics, 2006,Vol, 59, No. 5, pp. 355-363) and has posed worldwide major problems.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an enzyme incharge of the synthesis of lipid A (the hydrophobic anchor of LPS, whichis the constituent of the outer membrane).

Lipid A biosynthesis consists of reactions in 10 stages, and LpxCcatalyzes the second stage to remove the acetyl group ofUDP-3-O-acyl-N-acetylglucosamine (J. Biol. Chem., 1995, Vol. 270, pp.30384-30391). Lipid A is a component essential for the formation of theouter membrane, and is indispensable for the survival of Gram-negativebacteria (J. Bacteriol., 1987, Vol. 169, pp. 5408-5415). LpxC is one ofthe rate-determining important enzymes during the process of lipid Abiosynthesis, and is an indispensable enzyme for lipid A biosynthesis.Thus, a drug inhibiting the activity of LpxC is highly expected to becapable of becoming an antimicrobial agent effective againstGram-negative bacteria including Pseudomonas aeruginosa, particularlyagainst drug resistant Pseudomonas aeruginosa, because such a drug has amechanism of action different from those of conventional drugs.

Compounds having LpxC inhibitory activity have been known so far (PatentDocuments 1 to 7).

To provide its medicinal efficacy, a drug is required to dissolve at anabsorption site. Thus, when a sparingly water-soluble drug is orallyadministered, the drug may be insufficiently absorbed from thegastrointestinal tract and have a difficulty in providing its medicinalefficacy. Also, in the case of parenteral administration, particularlyintravenous administration, the drug is required to be administered in adissolved form.

Cyclodextrins (sometimes referred to as “CDs” hereinbelow), orcyclodextrin derivatives (sometimes referred to as “CD derivatives”hereinbelow), are known to be used for solubilizing compounds(International J. Pharmaceutics., 2013, Vol. 453, pp. 167-180; YakugakuZasshi, 2012, Vol. 132, No. 1, pp. 85-105).

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: International Publication No. WO 04/062601 pamphlet

Patent Document 2: International Publication No. WO 07/069020 pamphlet

Patent Document 3: International Publication No. WO 08/154642 pamphlet

Patent Document 4: International Publication No. WO 10/031750 pamphlet

Patent Document 5: International Publication No. WO 10/017060 pamphlet

Patent Document 6: International Publication No. WO 10/032147 pamphlet

Patent Document 7: International Publication No. WO 11/132712 pamphlet

SUMMARY OF INVENTION Problem to be Solved by the Invention

An object of the present invention is to provide a pharmaceuticalcomposition that exhibits potent antimicrobial activity againstGram-negative bacteria, including Pseudomonas aeruginosa and their drugresistant strains, by inhibiting LpxC, and has excellent watersolubility. Another object of the present invention is to provide amethod for producing an excellently stable liquid formulation comprisingthe pharmaceutical composition.

Means for Solving the Problem

Under such circumstances, the present inventors have intensively studiedto find that pharmaceutical compositions that contain hydroxamic acidderivatives selected from(2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethyny)benzoyl)(methyl)amino)-N-hydroxy-N′,2-dimethylmalonamide(sometimes referred to as “Compound A” hereinbelow),(2S)-2-((4-((4-((1R)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-hydroxy-N′,2-dimethylmalonamide(sometimes referred to as “Compound B” hereinbelow) and)(2S)-N-hydroxy-2-((4-((4-((1S)-1-hydroxy-2-methoxyethyl)phenyl)ethynyl)benzoyl)methly)amino-N′,2-dimethylmalonamide(sometimes referred to as “Compound C” hereinbelow), or salts thereof,and a solubilizing agent have potent antimicrobial activity andexcellent solubility and are useful as medicines. Additionally, theinventors have found that an excellently stable liquid formulation canbe produced by obtaining an aqueous solution of a hydroxamic acidderivative or a salt thereof and a solubilizing agent and then, asrequired, adjusting the pH of the obtained aqueous solution to 3 to 8,and thereby have completed the present invention.

That is, the present invention provides the following.

[1] A pharmaceutical composition comprising a hydroxamic acid derivativeselected from Compound A, Compound B and Compound C, or a salt thereof,and a solubilizing agent.[2] The pharmaceutical composition according to [1], wherein thesolubilizing agent is a CD or a CD derivative.[3]The pharmaceutical composition according to [1] or [2], wherein thehydroxamic acid derivative is Compound A.[4] The pharmaceutical composition according to [2]or [3], wherein theCD or the CD derivative is one or more selected from α-cyclodextrin(sometimes referred to as “αCD” hereinbelow), γ-cyclodextrin (sometimesreferred to as “γCD” hereinbelow), hydroxypropyl-α-cyclodextrin(sometimes referred to as “HPαCD” hereinbelow),sulfobutylether-β-cyclodextrin (sometimes referred to as “SBEβCD”hereinbelow), 2,3,6-tri-O-methyl-β-cyclodextrin,hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin (sometimesreferred to as “HPβCD” hereinbelow),heptakis-2,6-di-O-methyl-β-cyclodextrin (sometimes referred to as“DMβCD” hereinbelow), 6-O-α-maltosyl-β-cyclodextrin,methyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin (sometimesreferred to as “HPγCD” hereinbelow).[5] The pharmaceutical composition according to [2]or [3], wherein theCD or the CD derivative is one or more selected from αCD, γCD, HPαCD,SBEβCD, HPβCD and HPγCD.[6] The pharmaceutical composition according to any one of [1] to [5],wherein the pharmaceutical composition is a liquid formulation.

[7] The pharmaceutical composition according to [6], wherein a pH of theliquid formulation is from 3 to 8.

The pharmaceutical composition according to any one of [1] to [5],wherein the pharmaceutical composition is a frozen liquid formulation.[9] The pharmaceutical composition according to [8], wherein a pH of thefrozen liquid formulation when thawed is from 3 to 8.[10] The pharmaceutical composition according to any one of [1] to [5],wherein the pharmaceutical composition is a lyophilized formulation.[11] The pharmaceutical composition according to [10], wherein a pH ofan aqueous solution of the lyophilized formulation is from 3 to 8.[12] The pharmaceutical composition according to [1], wherein thesolubilizingagent is one or more selected from monoalcohols, polyhydricalcohols, amities, sulfoxides, amino acids, surfactants, acids andbases.[13] The pharmaceutical composition according to [12], wherein thehydroxamic acid derivative is Compound A.[14] The pharmaceutical composition according to [12] or [13], whereinthe monoalcohol is an alcohol having 1 to 6 carbon atoms; the polyhydricalcohol is a diol; and the acid is an organic acid.[15] The pharmaceutical composition according to [12] or [13], whereinthe solubilizing agent is one or more selected from alcohols having 1 to6 carbon atoms, diols, amino acids and organic acids in combination withan amide.[16] The pharmaceutical composition according to any one of [12] to[15], wherein the pharmaceutical composition is a liquid formulation.[17] The pharmaceutical composition according to any one of [1] to [16],wherein the pharmaceutical composition is a pharmaceutical compositionused as an LpxC inhibitor.[18] The pharmaceutical composition according to any one of [1] to [16],wherein the pharmaceutical composition is a pharmaceutical compositionused as an antimicrobial agent.

A method for producing a liquid formulation comprising a hydroxamic acidderivative or a salt thereof and a solubilizing agent, the methodcomprising: a step of dissolving the hydroxamic acid derivative selectedfrom Compound A, Compound B and Compound C or a salt thereof and thesolubilizing agent in water to obtain an aqueous solution of thehydroxamic acid derivative or the salt thereof, followed by adjusting apH of the obtained aqueous solution to from 3 to 8, as required.

[20] The production method according to [19], wherein the solubilizingagent is CD or a CD derivative.[21] The production method according to [19] or [20], wherein thehydroxamic acid derivative is Compound A.[22] The production method according to [20] or [21], wherein the CD orthe CD derivative is one or more selected from αCD, γCD, HPαCD, SBEβCD,2,3,6-tri-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, HPβCD,DMβCD, 6-O-β-maltosyl-β-cyclodextrin, methyl-β-cyclodextrin and HPγCD.[23] The production method according to [20] or [21], wherein the CD orthe CD derivative is one or more selected from αCD, γCD, HPαCD, SBEβCD,HPβCD and HPγCD.

The present invention also provides the following.

[24] The pharmaceutical composition according to any one of [1] to [11],further comprising a pH adjuster.[25] The pharmaceutical composition according to [24], wherein the pHadjuster is one or more selected from mineral acids, organic acids,inorganic bases and organic bases.[26] The pharmaceutical composition according to [25], wherein themineral acid is hydrochloric acid, sulfuric acid, phosphoric acid andnitric acid; the organic acid is maleic acid, benzoic acid, ascorbicacid, niethanesulfonic acid, acetic acid, malic acid, lactic acid,tartaric acid, citric acid, gluconic acid, glutamic acid, aspartic acidand adipic acid; the inorganic base is sodium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydrogen carbonate, sodiumdihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogenphosphate, dipotassium hydrogen phosphate, trisodium phosphate andammonia; and the organic base is disodium citrate, monoethanolamine,diethanolamine, trometamol, diisopropanoiamine, triethanolamine,triisopropanolamine, L-arginine, L-histidine, L-lysine and meglumine.[27] The pharmaceutical composition according to [25], wherein themineral acid is hydrochloric acid and sulfuric acid; the organic acid isacetic acid and citric acid; the inorganic base is sodium hydroxide andsodium carbonate; and the organic base is trometamol and meglumine.[28] The pharmaceutical composition according to any one of [24] to[27], wherein the pharmaceutical composition is a liquid formulation.[29] The pharmaceutical composition according to [28], wherein a pH ofthe liquid formulation is from 3 to 8.[30] The pharmaceutical composition according to any one of [24] to[27], wherein the pharmaceutical composition is a frozen liquidformulation.[31] The pharmaceutical composition according to [30], wherein a pH ofthe frozen liquid formulation when thawed is from 3 to 8.[32] The pharmaceutical composition according to any one of [24] to[27], wherein the pharmaceutical composition is a lyophilizedformulation.[33] The pharmaceutical composition according to [32], wherein a pH ofan injection formulation prepared from the lyophilized formulation isfrom 3 to 8.[34] The pharmaceutical composition according to any one of [24] to[33], wherein the pharmaceutical composition is a pharmaceuticalcomposition used as an LpxC inhibitor.[35] The pharmaceutical composition according to any one of [24] to[33], wherein the pharmaceutical composition is a pharmaceuticalcomposition used as an antimicrobial agent.[36] The pharmaceutical composition according to any one of [1] to [16]and [24] to [33], wherein the pharmaceutical composition is apharmaceutical composition used for treatment of Gram-negative bacterialinfection.

ADVANTAGEOUS EFFECTS OF THE INVENTION

The pharmaceutical composition comprising the hydroxamic acid derivativeof the present invention or a salt thereof and a solubilizina agentexhibits potent antimicrobial activity, has excellent water solubility,and is useful as a medicine. Furthermore, the production methodaccording to the present invention is useful as a method for producing aliquid formulation comprising a pharmaceutical composition havingexcellent stability.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail below.

“%” means herein “% by mass,” unless otherwise indicated.

Treatment means prophylaxis, therapy or the like against diseases.

<Hydroxamic acid Derivative>

Examples of the hydroxamic acid derivative include hydroxamic acidderivatives selected from Compound A, Compound B and Compound C, andCompound A is preferred.

The hydroxamic acid derivative can be produced in accordance with, forexample, Production Examples described below.

When the hydroxamic acid derivative or a salt thereof has isomers (forexample, optical isomers, geometrical isomers and tautomers), thepresent invention encompasses these isomers and also encompasses theirsolvates, hydrates, and crystals of various forms.

Examples of the salt of the hydroxamic acid derivative include saltswith alkali metal, such as sodium and potassium; salts with alkalineearth metal, such as calcium and magnesium; ammonium salts; and saltswith nitrogen-containing organic bases, such as trimethylamine,triethylamine, tributylamine, pyridine, N,N-dimethylaniline,N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine,dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine andN,N′-dibenzylethylenediamine.

Preferred salts among the salts described above includepharmacologically acceptable salts.

Administration methods, doses and frequency of administration of thehydroxamic acid derivative or a salt thereof may be selected asappropriate depending on the age, body weight and condition of apatient. Usually for adults, it may be orally or parenterally (forexample, by injection, infusion, or administration to the rectal site)administered in an amount of 001 to 1000 mg/kg/day in one to severalportions.

<Solubilizing Agent>

In the present invention, a solubilizing agent can be used to achieveexcellent solubility of the hydroxamic acid derivative or a saltthereof.

Examples of the solubilizing agents include monoalcohols, polyhydricalcohols, amides, sulfoxides, amino acids, surfactants, acids, bases,CDs and CD derivatives,

Examples of the monoalcohols include alcohols having 1 to 6 carbonatoms, such as ethanol, propanol, 2-propanol, butanol and chlorobutanol;alcohols having 7 to 20 carbon atoms, such as 3-indolepropanol, benzylalcohol, octanol, nonanol, decanol, undecyl alcohol, lauryl alcohol,tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol,heptadecyl alcohol and stearyl alcohol; and unsaturated alcohols having7 to 20 carbon atoms, such as oleyl alcohol, linoleyl alcohol andlinolenyl alcohol.

Preferred monoalcohols include alcohols having 1 to 6 carbon atoms andalcohols having 7 to 20 carbon atoms. Alcohols having 1 to 6 carbonatoms and benzyl alcohol are preferred, ethanol, propanol, 2-propanol,butanol and benzyl alcohol are more preferred, ethanol and benzylalcohol are further preferred, and ethanol is particularly preferred.

Examples of the polyhydric alcohols include diols, such as ethyleneglycol, propylene glycol, 1,3-propanediol, 1,2-butanediol,1,3-butanediel, 1,4-butanediel, 2,3-butanediol, 1,5-pentanediol,dipropylene glycol, diethylene glycol triethylene polyethylene glycol300, polyethylene glycol 400, polyethylene glycol 600, polyethyleneglycol 1500, polyethylene glycol 4000 and alpha-thioglycerin; andtriols, such as glycerin.

Preferred polyhydric alcohols include diols. Propylene glycol,1,3-butanediol, dipropylene glycol, diethylene glycol, triethyleneglycol, polyethylene glycol 300, polyethylene glycol 400, polyethyleneglycol 600, polyethylene glycol 1500 and polyethylene glycol 4000 aremore preferred, and triethylene glycol and polyethylene glycol 400 arefurther preferred.

Examples of the amides include N,N-dimethylacetainide, polyvinylpyrrolidone, urea, ethyl urea and nicotinic acid amide.

Preferred amides include N,N-dimethylacetamide, urea, ethyl urea andnicotinic acid amide. N,N-dimethylacetamide and nicotinic acid amide aremore preferred.

Examples of the sulfoxides include dimethyl sulfoxide.

Examples of the amino acids include amino acids, such as glycine,alanine, β-alanine, valine, leucine, isoleucine, serine, threonine,cysteine, rnethionine, aspartic acid, glutamic acid, asparagine,glutamine, arginine, lysine, histidine, hydroxylysine, phenylalanine,tyrosine, tryptophan, N-acetyltryptophan, proline, hydroxyproline,cystine, L-glutamic acid L-lysine, taurine, β-alanine tert-butyl esterand phenylalanine tent-butyl ester, derivatives thereof, and saltsthereof.

Preferred amino acids include glycine, alanine, β-alanine, asparticacid, glutamic acid, arginine, lysine, histidine, hydroxylysine,phenylalanine, tryptophan, N-acetyltryptophan, proline, hydroxyproline,taurine, β-alanine tent-butyl ester and phenylalanine tert-butyl ester.β-Alanine, arginine, histidine, phenylalanine, tryptophan,N-acetyltryptophan, proline, taurine, β-alanine tert-hutyl ester andphenylalanine tert-butyl ester are more preferred, and β-alanine,phenylalanine and tryptophan are further preferred.

Examples of the surfactants include sodium lauryl sulfate, dioctylsodium sulfosuccinate, polysorbates, polyoxyethylene hydrogenated castoroil, Cremophor and sucrose fatty acid esters.

Preferred surfactants include polysorbates, polyoxyethylene hydrogenatedcastor oil, Cremophor and sucrose fatty acid esters.

Examples of the acids include organic acids, such as maleic acid,benzoic acid, ascorbic acid, methanesulfonic acid, acetic acid, malicacid, lactic acid, tartaric acid, citric acid, gluconic acid, adipicacid, succinic acid, thioglycolic acid, deoxycholic acid,ursodeoxycholic acid, edetic acid, salicylic acid, meta-sulfobenzoicacid, cinnamic acid, 3-phenylpropionic acid,3-(4-hydroxyphenyl)propionic acid, besylic acid, p-toluenesulfonic acid,sugar acid and chondroitin sulfate; saturated fatty acids having 8 to 20carbon atoms, such as caprylic acid, pelargonic acid, capric acid,undecylic acid, lauric acid, tridecylic acid, myristic acid,pentadecylic acid, palmitic acid, margaric acid and stearic acid;unsaturated fatty acids having 8 to 20 carbon atoms, such as citronellicacid, undecylenic acid, linderic acid, physeteric acid, zoomaric acid,palmitoleic acid, oleic acid and linoleic acid; and inorganic acids,such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid,sodium dihydrogen phosphate and potassium dihydrogen phosphate.

Preferred acids include organic acids. Maleic acid, benzoic acid,ascorbic acid, methanesulfonic acid, acetic acid, lactic acid, tartaricacid, citric acid, gluconic acid, adipic acid, thioglycolic acid,deoxycholic acid, ursodeoxycholic acid, salicylic acid,meta-sulfobenzoic acid, cinnamic acid, 3-phenylpropionic acid,3-(4-hvdroxyphenyl)propionic acid, besylic acid and p-toluenesulfonicacid are more preferred, benzoic acid, citric acid, cinnamic acid,3-phenylpropionic acid, 3-(4-hydroxyphenyl)propionic acid andp-toluenesulfonic acid are further preferred, and benzoic acid isparticularly preferred.

Examples of the bases include salts of organic acids, such as disodiumcitrate, sodium citrate, sodium acetate, sodium lactate, magnesiumgluconate, calcium saccharate and sodium benzoate; organic bases, suchas monoethanolamine, diethanolamine, trometamol, diisopropanolamine,triethanolamine, triisopropanolamine, ethylenediamine and meglumine; andinomanic bases, such as sodium hydroxide, potassium hydroxide, sodiumcarbonate, potassium carbonate, sodium hydrogen carbonate, disodiumhydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphateand ammonia.

Preferred bases include disodium citrate, sodium citrate, sodiumacetate, sodium lactate, magnesium gluconate, calcium saccharate, sodiumbenzoate, diethanolamine, trometamol, diisopropanolamine,triethanolamine, ethylenediamine, meglumine, sodium hydroxide, sodiumcarbonate, sodium hydrogen carbonate, disodium hydrogen phosphate andtrisodium phosphate. Sodium citrate, sodium lactate, sodium benzoate,trometamol, meglumine and sodium hydroxide are more preferred.

Examples of the CDs include αCD, β-cyclodextrin (sometimes referred toas “βCD” hereinbelow), or γCD.

Examples of the CD derivative include HPαCD, dimethyl-β-cyclodextrin,SBEβCD, 2,3,6-triacetyl-β-cyclodextrin,2,3,6-tri-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, HPβCD,DMβCD, 6-O-β-maltosyl-β-cyclodextrin, methyl-β-cyclodextrin,monoacetyl-β-cyclodextrin, monochlorotriazino-β-cyclodextrin, or HPγCD.

Preferred CDs or CD derivatives include αCD, γCD, HPαCD, SBEβCD,2,3,6-tri-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, HPβCD,DmβCD, 6-O-α- maltosyl-β-cyclodextrin, methyl-β-cyclodextrin, or HPγCD.αCD, γCD, HPαCD, SBEβCD, HPβCD, or HPγCD is more preferred.

Preferred solubilizing agents of the present invention include CDs andCD derivatives.

Additionally, preferred solubilizing agents of the present inventioninclude one or more selected from monoalcohols, polyhydric alcohols,amities, sulfoxides, amino acids, surfactants, acids and bases. Acombination of one or more selected from alcohols having 1 to 6 carbonatoms, diols, amino acids and organic acids with an amide is morepreferred.

The pharmaceutical composition of the present invention is provided as aliquid formulation, a frozen liquid formulation, or a lyophilizedformulation.

Subsequently, the method for producing the pharmaceutical composition ofthe present invention will be described.

Production Method 1 Liquid formulation

The liquid formulation can be produced by dissolving a hydroxamic acidderivative or a salt thereof and a solubilizing agent in water.

The hydroxamic acid derivative is preferably Compound A.

The solubilizing agent is preferably a CD or a CD derivative.

The amount of the solubilizing agent should be sufficient to dissolvethe hydroxamic acid derivative or the salt thereof. The amount may beusually 1 to 20 molar times, and is preferably 1.2 to 10 molar times,and more preferably 1.5 to 5 molar times, relative to the hydroxamicacid derivative or the salt thereof.

In another aspect, the preferred solubilizing agent is one or moreselected from monoalcohols, polyhydric alcohols, amides, sulfoxides,amino acids, surfactants, acids and bases.

It should be noted that sterilization treatment and the like in theproduction of the liquid formulation of the present invention may beconducted in accordance with the procedures usually performed.

The pH of a liquid formulation of the present invention is preferablyfrom 3 to 8, more preferably from 3.5 to 7.5, and further preferablyfrom 4.0 to 6.5.

The pH of the liquid formulation is preferably adjusted to from 3 to 8,more preferably to from 3.5 to 7.5, and further preferably to from 4.0to 6.5 by adding a pH adjuster as required.

Examples of the pH adjuster used include one or more selected frommineral acids, organic acids, inorganic bases and organic bases.

Examples of the mineral acid used as the pH adjuster includehydrochloric acid, sulfuric acid, phosphoric acid and nitric acid.Hydrochloric acid, sulfuric acid and phosphoric acid are preferred, andhydrochloric acid and sulfuric acid are more preferred.

Examples of the organic acid used as the pH adjuster include maleicacid, benzoic acid, ascorbic acid, methanesulfonic acid, acetic acid,malic acid, lactic acid, tartaric acid, citric acid, gluconic acid,glutamic acid, aspartic acid and adipic acid. Ascorbic acid,methanesulfonic acid, acetic acid, citric acid and aspartic acid arepreferred, and acetic acid and citric acid are more preferred.

Examples of the inorganic base used as the pH adjuster include sodiumhydroxide, potassium hydroxide, sodium carbonate, sodium hydrogencarbonate, sodium dihydrogen phosphate, potassium dihydrogen phosphate,disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodiumphosphate and ammonia. Sodium hydroxide, sodium carbonate, sodiumhydrogen carbonate and tri sodium phosphate are preferred, and sodiumhydroxide and sodium carbonate are more preferred.

Examples of the organic base used as the pH adjuster include disodiumcitrate, monoethanolamine, diethanolamine, trometamol,diisopropanolarnine, triethanolamine, triisopropanolamine, L-arginine,L-histidine, L-lysine and meglumine. Monoethanolamine, diethanolamine,trometamol, triethanolamine and meglumine are preferred, and trometamoland meglumine are more preferred.

Additives usually used, such as osmo-regulators, stabilizers,surfactants, soothing agents, excipients and/or preservatives, may beadded to the liquid formulation of the present invention, as required.

Examples of the osmo-regulator include glucose, sodium chloride,D-mannitol, glycerin and propylene glycol.

Examples of the stabilizer include sodium hydrogen sulfite, sodiummetabisulfite, potassium metabisulfite, sodium pyrophosphate, sodiumthiosulfate, sodium metasulfobenzoate, sodium formaldehydesulfoxylate,ethylenediamine, disodium edetate, thioglycolic acid, sodium gluconate,potassium L-glutamate, L-lysine-L-glutamate, chondroitin sulfate sodium,L-aspartic acid, L-cysteine and dibutylhydroxytoluene.

Examples of the surfactant include sorbitan fatty acid esters,polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fattyacid esters and polyoxyethylene-polyoxypropylene glycol copolymers.

Examples of the soothing agent include lidocaine, procaine, meprylcaineand benzyl alcohol.

Examples of the excipient include sugars such as trehalose, maltose,glucose, lactose, sucrose and fructose, sugar alcohols such asD-sorbitol, xylitol, inositol and D-mannitol, or amino acids such asglycine, L-alanine, L-phenylalanine, L-leucine, L-isoleucine, taurine,DL-methionine, L-serine, L-threonine, L-glutamine, sodium L-glutamate,acetyltryptophan and L-histidine.

Examples of the preservative include methyl parahydroxybenzoate, ethylparahydroxybenzoate, propyl parahydroxybenzoate, butylparahydroxybenzoate, disodium edetate, tetrasodium edetate,chlorobutanol, chlorhexidine gluconate, benzalkonium chloride andhenzethonium chloride.

The liquid formulation of the present invention can be provided as aninjection liquid formulation.

The content of the hydroxamic acid derivative in the liquid formulationof the present invention is preferably from Ito 100 mg/mL, morepreferably from 2 to 50 mg/mL.

The dose of the hyd.roxamic acid derivative is determined asappropriate, depending on usages, the age and sex of the patient,disease forms, other conditions, and the like.

Usually, the derivative may be administered in an amount of 0.1 to 1000mg/kg/day to an adult.

Production Method 2 Frozen Liquid Formulation

The frozen liquid formulation can be produced by freezing the liquidformulation obtained by the method described in Production method 1.

The temperature of the freezing step should be temperatures at which theliquid formulation can be frozen, and the temperature is preferably from−78 to −15° C.

The time period of the freezing step may be, but not particularlylimited to, from 1 to 24 hours.

It should be noted that sterilization treatment and the like in theproduction of the frozen liquid formulation of the present invention maybe conducted in accordance with the procedures usually performed.

The frozen liquid formulation of the present invention can be thawed tobe provided as an injection liquid formulation.

The pH of the frozen liquid formulation when thawed is preferably from 3to 8, more preferably from 3.5 to 7.5, and further preferably from 4.0to 6.5.

The content of the hydroxamic acid derivative in the frozen liquidformulation when thawed is preferably froml to 100 mg/mL, morepreferably from 2 to 50 mg/mL.

The dose of the hydroxamic acid derivative is determined as appropriate,depending on usages, the age and sex of the patient, disease forms,other conditions, and the like. Usually, the derivative may beadministered in an amount of 0.1 to 1000 mg/kg/day to an adult.

Production Method 3 Lyophilized Formulation

The lyophilized formulation can be provided by lyophilizing the liquidformulation obtained by the method described in Production method 1.

This step may be conducted in accordance with a lyophilized methodusually conducted. For example, the step can be conducted in accordancewith “15.2 Touketsu kansou no jissai (Practical lyophilization)”described in “Iyakuhin no jissai (Practical pharmaceuticals),” Vol. 11,Seizai no tanisousa to kikai (Unit operation and machines forpharmaceutica edited by Yoshinobu Nakai, pp. 388 to 396 (1988, HirokawaShoten Co.).

To the lyophilized formulation of the present invention, additives canbe added to improve the solubility and/or appearance.

Examples of the additive include amino acids, polyethylene glycols,sugars, sugar alcohols, urea, ethyl urea, creatinine, trometamol,purified soya lecitin and polysorbate 80. These can be used singly or inmixture of two or more.

Preferred additives include amino acids, polyethylene glycols, sugarsand sugar alcohols. Amino acids, sugars and sugar alcohols are morepreferred, and sugar alcohols are further preferred.

Examples of the amino acid used as an additive include glycine,L-alanine, phenylalanine, L-valine, L-leucine, L-isoleucine, taurine,DL-methionine, L-serine, L-threonine, L-glutamine, sodium L-glutamate,acetyltryptophan and L-histidine. Glycine, L-serine, threonine,L-alanine, L-leucine and L-isoleucine are preferred, and glycine is morepreferred.

Examples of the polyethylene glycol used as an additive includepolyethylene glycol 300, polyethylene glycol 400, polyethylene glycol600, polyethylene glycol 4000 and polyethylene glycol 6000. Polyethyleneglycol 300, polyethylene glycol 600 and polyethylene glycol 4000 arepreferred.

Examples of the sugar used as an additive include trehalose, maltose,glucose, lactose, sucrose, fructose and dextran. Trehalose, glucose,sucrose and fructose are preferred.

Examples of the sugar alcohol used as an additive include D-sorbitol,xylitol, inositol and D-mannitol. D-sorbitol, xylitol and D-mannitol arepreferred.

In the production of the lyophilized formulation of the presentinvention, the sterilization treatment and the like may be conducted inaccordance with the procedures usually performed.

The lyophilized formulation of the present invention can be dissolvedwith water for injection and the like to be provided as an injectionformulation.

The pH of the injection formulation prepared from the lyophilizedformulation is preferably from 3 to 8, more preferably from 3.5 to 7.5,and further preferably from 4.0 to 6.5.

The content of the hydroxamic acid derivative in the injectionformulation prepared from the lyophilized formulation is preferably from1 to 100 mg/mL, more preferably from 2 to 50 mg/mL.

The dose of the hydroxamic acid derivative is determined as appropriate,depending on usages, the age and sex of the patient, disease forms,other conditions, and the like. Usually, the derivative may beadministered in an amount of 0.1 to 1000 mg/kg/day to an adult.

The liquid formulation, frozen liquid formulation and lyophilizedformulation of the present invention are preferably produced withoutusing organic solvent. Thus, these formulations contain no residualsolvent and are safe for human bodies.

The present invention will be described referring to ProductionExamples, Examples and Test Examples, but the present invention is notintended to be limited to these.

Unless specifically mentioned, the silica gel column chromatography isflash column chromatography, and its carrier is B. W. silica gel BW-300,Fuji Silysia Chemical Ltd.

The mixture ratio in the eluant is the volume ratio.

The conditions of lyophilization are as follows.

A vial is cooled to −60° C. to freeze its content. Then, the shelftemperature is increased to −10° C. in vacuo (50 Pa or less), andprimary drying is carried out at the same pressure and temperature. Whenthe product temperature has reached −10° C. or more, the shelftemperature is increased to 20° C., and secondary drying is carried outat the same pressure and temperature. The drying is considered to becompleted when the product temperature approximately corresponds to thesetting temperature and exhibits no change.

Each abbreviation has the following meaning.

DMSO-d₆: Heavy dimethyl sulfoxideESI: Electrospray ionizationIPE: Diisopropyl ether

Me: Methyl

TBS: tert-Butyldimethylsilyl

THP: Tetrahydro-2H-pyran-2-yl s: Singlet d: Doublet

dd: Double doublet

m: Multiplet

In a ¹H-NMR spectrum, for example, the description of [1.81], 1.82 (3H,s) indicates that peaks derived from each diastereomer in a diastereomermixture are observed at 1.81 and 1.82 as a singlet, and the total numberof protons is 3H.

Production Example 1

To 1000 mL of N-methylpyrrolidone, 421 g of N-methylbenzylamine and 400g of diethyl 2-bromo-2-methylmalonate were added and stirred at 100° C.for an hour. Then, the reaction mixture was cooled. After 1.5 L oftoluene and 1.5 L of water were added sequentially, 70 mL ofhydrochloric acid was added. The organic layer was separated, and thesolvent was distilled off under reduced pressure to obtain 499 g of acolorless oily product.

To 400 g of the obtained oily product, 2.0 L of ethyl acetate, 32 g of10% palladium on carbon (50% wet) and 81.9 g of acetic acid were addedsequentially and stirred under hydrogen atmosphere (0.5 MPa) at 45° C.for 18 hours and 30 minutes. After the reaction mixture was cooled andfiltered over celite, the residue was washed with 400 mL of ethylacetate. To the filtrate, 1200 mL of water was added. Hydrochloric acidwas used to adjust the pH to 2 or less, and the aqueous layer wasseparated. To the obtained aqueous layer, 1200 mL of ethyl acetate wasadded, and a 20% sodium hydroxide aqueous solution was used to adjustthe pH to 9. The organic layer was separated and the solvent wasdistilled off under reduced pressure to obtain 204 g of a colorless oilyproduct.

To 200 g of the obtained oily product, 1.0 L of acetonitrile and 198 gof sodium hydrogen carbonate were added. Then, 168 g of benzylchloroformate was added dropwise under ice cooling over 25 minutes. Thereaction mixture was warmed to room temperature, stirred for 7 hours and45 minutes, and allowed to stand overnight. Then, the reaction mixturewas stirred at 40 to 45° C. for 1 hour and 30 minutes, and cooled, andthen, an insoluble material was filtered off. The residue was washedwith 200 mL of acetonitrile. The filtrate and the washed solution werecombined and concentrated under reduced pressure to obtain 324 g of acolorless oily product.

To 1968 mL of water, 15.36 g of sodium dihydrogen phosphate dihydratewas added, and 1125 mL, of a 0.05 mol/L sodium hydroxide aqueoussolution was added. To this aqueous solution, a mixture of 120 g of theobtained oily product and 360 mL of acetonitrile was added at 24° C.,stirred at the same temperature for 2 hours and 45 minutes, and then,allowed to stand overnight. Additionally, the reaction mixture wasstirred at the same temperature for 6 hours, and then, allowed to standfor 22 hours. Subsequently, 30 mL of a 0.05 mol/L phosphate buffersolution (pH 7.4) was added to 2.9 g (20 units/mg) of porcine liveresterase and subjected to ultrasonic irradiation for 30 minutes toprovide a suspension solution, which was added to the reaction mixtureat 25° C. The reaction mixture, of which pH was adjusted with a 1 mol/Lsodium hydroxide aqueous solution within the range of 6.7 to 7.1, wasstirred at 26° C. for 5 hours. To the reaction mixture, 1200 mL of ethylacetate was added, and 37 mL of hydrochloric acid, 300 g of sodiumchloride and 48 g of Celpure were added sequentially under ice cooling.After stirring at the same temperature for an hour, an insolublematerial was filtered off. The residue was washed with 240 mL, of ethylacetate, and the filtrate and the washed solution were combined. Theorganic layer was separated, and the aqueous layer was extracted with180 mL of ethyl acetate. The organic layer and the extract solution werecombined, and 48 g of anhydrous sodi urn sulfate and 1.2 g of activatedcarbon were added. After stirring for 30 minutes, the mixture wasfiltered over celite. The residue was washed with 180 mL of ethylacetate, and the filtrate and the washed solution were combined. Then,1540 mL of the solvent was distilled off under reduced pressure. To theobtained residue, 240 mL of heptane was added and cooled to 18° C. over2 hours. The solid was filtered off and washed with 120 mL, of heptanetwice to obtain 88.18 g (>99.9% ee) of(((((2R)-2-carboxy-1-ethoxy-1-oxopropan-2-yl)(methyl)carbamoyl)oxy)methyl)benzeneas colorless crystals.

1H-NMR (400 MHz, DMSO-d6)δ:1.15-1.20 (3H, m), 1.61 (3H, s), 2.86 (3E1,s), 3.95-4.15 (2H, m), 5.07 (2H, s), 7.28-7.43 (5H, m)

HPLC Measurement Conditions

Column: 4.6×150 mm CHIRALPAK IA 5 μm

Measurement wavelength: 210 nm

Column temperature: 40° C.

Mobile phase: hexane:ethanol=95:5 (0.1% trifluoroacetic acid)

Flow rate: 0.7 mL/minute

Production Example 2

To 250 g of(((((2R)-2-carboxy-1-ethoxy-1-oxopropan-2-yl)(methyl)carbamoy)oxy)methyl)benzene,1300 mL of ethyl acetate and 1.0 mL of N,N-dimethylformamide were added.After 133 g of oxalyl chloride was added dropwise at 5° C. over 20minutes, 200 mL of ethyl acetate was added. The reaction mixture waswarmed to 20° C. and stirred for 4 hours. Under reduced pressure, 1395mL of the solvent was distilled off. To the obtained residue, 1000 mL oftetrahydrofuran was added and cooled to 8° C. At the same temperature,94.1 g of triethylamine and 109 g ofO-(tetrahydro-2H-pyran-2-yl)hydroxylamine were added sequentially, andwarmed to 20° C. over 3 hours with stirring. After the reaction mixturewas allowed to stand overnight, 225 mL of acetone was added and stirredfor 40 minutes. Subsequently, 750 mL of toluene and 1000 mL water wereadded and cooled to 10° C. Then 62 mL of hydrochloric acid was added.Additionally, the pH was adjusted to 3 with 6 moL/L hydrochloric acidand a 20% sodium hydroxide aqueous solution, and the aqueous layer wasseparated. To the obtained aqueous layer, 1250 mL of ethyl acetate wasadded, and 210 mL of a 20% sodium hydroxide aqueous solution was added.Subsequently, 1450 g of sodium chloride was added, and the resultantsolution was warmed to 30° C. The organic layer was separated, and theaqueous layer was extracted with 750 mL of ethyl acetate. The organiclayer and the extract solution were combined, and the solvent wasdistilled off under reduced pressure. To the obtained residue, 250 mL oftoluene was added. The solvent was distilled off under reduced pressureto obtain 215 g of an orange oily product.

To 213 g of the obtained oily product, a 40% methylamine/methanolsolution was added at room temperature, stirred at 40 to 43° C. for 8hours and 30 minutes, and then, allowed to stand overnight.Additionally, after stirring at 45° C. for 5 hours, the solvent wasdistilled off under reduced pressure. To the obtained residue, toluenewas added, and the solvent was distilled Wunder reduced pressure.Subsequently, tetrahydrofuran was added to the obtained residue, and thesolvent was distilled off under reduced pressure to obtain 203 g of ayellow oily product.

To 203 g of the obtained oily product, 1400 mL of tetrahydrofuran wasadded, and 117 g of sodium hydrogen carbonate was added at 35° C.Subsequently, a mixture of 168 g of 4-iodobenzoyl chloride and 200 ml,of tetrahydrofuran, and 100 mL of tetrahydrofuran were added at the sametemperature and stirred for 5 hours. After 58.2 g of sodium hydrogencarbonate and 29 mL of morpholine were added to the reaction mixture atthe same temperature and stirred for 2 hours, the mixture was allowed tostand overnight at room temperature. To the reaction mixture, 1370 mL ofethyl acetate, 1700 mL of water and 170 g of sodium chloride were addedsequentially, and the organic layer was separated. After 860 mL of waterand 42.7 g of sodium chloride were added to the obtained organic layerand stirred for 15 minutes, the organic layer was separated. Theobtained organic layer was filtered, and the solvent of the filtrate wasdistilled off under reduced pressure. After 300 mL of ethyl acetate and300 mL of toluene were added to the obtained residue and stirred at 30°C. for an hour, the mixture was allowed to stand overnight. The solidwas filtered off and washed with an ethyl acetate/toluene mixture (1:1,300 mL) to obtain 206 g of a brown solid. After 2000 mL of ethyl acetatewas added to the obtained brown solid and stirred at 40° C. for an hour,the mixture was cooled under ice cooling, and the solid was filteredoff. The solid was washed with ethyl acetate to obtain 148.9 g (>99.9%ee) of(2S)-2-((4-iodobenzoyl)(methyl)amino)-N,2-dimethyl-N′-(tetrahydro-2H-pyran-2-yloxy)malonamideas colorless crystals.

¹H-NMR (400 MHz, DMSO-d6)δ: 1.40-1.75 (6H, m), 1.61 (3H, s), [2.62] 2.63(3H, d, J=3.7 Hz), 2.99 (3H, d, J=2.7 Hz), 3.40-3.60 (1H, m),[3.82-3.92] 3.92-4.02 (1H, m), [4.74-4.80] 4.80-4.86 (1H, m), [7.31]7.33 (2H, d, J=8.2 Hz), 7.85 (2H, d, J=8.3 Hz), [8.25-8.33] 8.35-8.43(1H, m), 11.52 (1H, s)

HPLC Measurement Conditions

Column: 4.6×250 mm CHIRALPAK ID 5 μm

Measurement wavelength: 230 nm

Column temperature: 40° C.

Mobile phase: hexane:ethanol 85:15

Flow rate: 1.0 mL/minute

Production Example 3

To a mixture of 1.08 g of (1S)-1-(4-bromophenyl)ethane-1,2-diol, 350 mgof bis-triphenylphosphinepalladium(II) dichloride, 190 mg of copper(I)iodide, and 10 mL of n-butyl acetate, 7.8 mL oftriisopropylsilylacetylene and 7.0 mL of triethylamine were added undera nitrogen atmosphere, and the resulting mixture was stirred underreflux for 1 hour. The reaction mixture was cooled, a saturated aqueoussolution of ammonium chloride was added, the pH was adjusted to 6.2 with6 mol/L hydrochloric acid, then Celpure and ethyl acetate were added,and then the insoluble material was filtered off. The organic layer ofthe filtrate was separated, washed with a saturated aqueous solution ofsodium chloride, and then dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the obtainedresidue was subjected to silica gel column chromatography [eluent; ethylacetate:hexane=40:60→45:55] to obtain 1.32 g of a yellow oil.

To a mixture of 1.32 g of the obtained yellow oil and 13 mL oftetrahydrofuran, 6.2 mL of a 1 mol/L solution of tetra-n-butylamtnoniumfluoride in tetrahydrofuran was added under ice cooling, and theresulting mixture was stirred at the same temperature for 30 minutes andthen at room temperature for 45 minutes. A saturated aqueous solution ofammonium chloride was added to the reaction mixture, the pH was adjustedto 2.0 with 1 mol/L hydrochloric acid, and then ethyl acetate was added.The organic layer was separated, and the aqueous layer was extractedwith ethyl acetate twice. The organic layer was combined with theextract, washed with a saturated aqueous solution of sodium chloride,and then dried over anhydrous magnesium sulfate. The solvent wasdistilled off under reduced pressure, and the obtained residue waspurified by silica gel column chromatography [eluent; ethylacetate:hexane=50:50→70:30] to obtain 513 mg of a light brown solid.Hexane was added thereto, and the solid material was collected byfiltration to obtain 466 mg of (1S)-1-(4-ethynylphenyl)ethane-1,2-diolas a light brown solid.

¹H-NMR (400 MHz, CDCl₃)δ:1.97-2.07 m), 2.56 (1H, d, J=3.4 Hz), 3.08 (1H,s), 3.56-3.70 (1H, m), 3.71-3.82 (1H, m), 4.79-4.88 (1H, m), 7.34 (2H,d, J=8.3 Hz), 7.49 (2H, d, J=8.3 Hz).

Production Example 4

To a mixture of 587 mg of(2S)-2-((4-iodobenzoyl)(methyl)amino)-N,2-dimethyl-N′-(tetrahydro-2H-pyran-2-yloxy)malonamide,253 mg of (1S)-1-(4-ethynylphenyl)ethane-1,2-diol, 84 mg ofbis-triphenylphosphinepalladium(II) dichloride, 46 mg of copper(I)iodide, and 6.0 mL of tetrahydrofuran, 0.59 mL of triethylamine wasadded under a nitrogen atmosphere and under ice cooling, and theresulting mixture was stirred at the same temperature for 2 hours. Asaturated aqueous solution of ammonium chloride and ethyl acetate wereadded to the reaction mixture, and the pH was adjusted to 6.4 with 1mol/L hydrochloric acid. The organic layer was separated, and theaqueous layer was extracted with ethyl acetate. The organic layer wascombined with the extract, washed with a saturated aqueous solution ofsodium chloride, and then dried over anhydrous sodium sulfate. Thesolvent was distilled off under reduced pressure, and the obtainedresidue was purified by silica gel column chromatography [eluent;acetone:chloroform=40:60] to obtain 767 mg of(2S)-2-((4-((4-((1S)-1,2-dihydroxyethy)phenyl)ethynyl)benzoyl)(methyl)amino)-N,2-dimethyl-N′-(tetrahydro-2H-pyran-2-yloxy)malonamide as a pale yellowfoamy solid.

¹H-NMR (400 MHz, CDCl₃)δ: 1.50-1.68 (3H, m), 1.71-1.92 (3H, m), [1.82],1.83 (3H, s), 2.08-2.14 (1H, m), 2.63-2.68 (1H, m), [2.86], 2.87 (3H, d,J=4.1 Hz), [3.17], 3.20 (3H, s), 3.53-3.83 (3H, m), 3.83-4.07 (1H, m),4.83-4.89 (1H, m), 4.93-5.03 (1H, m), 7.37 (2H, d, J=8.0 Hz), 7.48-7.61(6H, m), [6.97-7.04], 7.61-7.67 (1H, m), [10.10], 10.51 (1H, s)

Production Example 5

To a mixture of 767 mg of(2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N,2-dimethyl-N′-(tetrahydro-2H-pyran-2-yloxy)malonamide and 6.0 mL of methanol, 46 mg ofp-toluenesulfonic acid monohydrate was added under ice cooling, and theresulting mixture was stirred at the same temperature for 40 minutes andthen at room temperature for 1 hour. Water and ethyl acetate were addedto the reaction mixture, and the resulting mixture was neutralized witha saturated aqueous solution of sodium hydrogen carbonate. The organiclayer was separated, ethyl acetate and sodium chloride were added to theaqueous layer, and the solid material was collected by filtration. Theorganic layer of the filtrate was separated, ethyl acetate and sodiumchloride were added to the aqueous layer, and the solid material wascollected by filtration. The organic layer of the filtrate wasseparated, the organic layer and the solid material thus obtained werecombined together, and then the solvent was distilled off under reducedpressure. The obtained residue was purified by silica gel columnchromatography [eluent; methanol:chloroform=10:90→15:85] to obtain 585mg of a yellow foamy solid. Ethyl acetate and IPE were added thereto,and the solid material was collected by filtration to obtain 463 mg of(2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methy)amino)-N-hydroxy-N′,2 -dimethyl-malonamide (Compound A) as a yellow solid.

¹H-NMR (400 MHz, CD₃OD)δ: 1.77 (3H, s), 2.79 (3H, s), 3.17 (3H, s),3.55-3.68 (2H, m), 4.67-4.74 (1H, m), 7.41 (2H, d, J=8.3 Hz), 7.51 (2H,d, J=8.3 Hz), 7.55 (2H, d, J=8.5 Hz), 7.68 (2H, d, J=8.5 Hz); MS (ESI):462[M+Na]⁺, 438[M−H]⁻

Production Example 6

In the same manner as in Production Example 3, from 1.09 g of(1R)-1-(4-bromophenyl)ethane-1,2-diol, 558 mg of(1R)-1-(4-ethynylphenyl)ethane-1,2-diol was obtained as a white solid.

1H-NMR (400 MHz, CDCl₃)δ:2.00 (1H, dd, J=7.1, 4.9 Hz), 2.54 (1H, d,J=3.4 Hz), 3.08 (1H, s), 3.60-3.68 (1H, m), 3.73-3.81 (1H, m), 4.80-4.88(1H, m), 7.34 (2H, d, J=8.1 Hz), 7.49 (2H, d, J=8.0 Hz)

Production Example 7

In the same manner as in Production Example 4, from 587 mg of(2S)-2-((4-iodobenzoyl)(methy)amino)-N,2-dimethyl-N-(tetrahydro-2H-pyran-2-yloxy)malonamide and 291 mg of (1R)-1-(4-ethynylphenyl)ethane-1,2-diol,797 mg of (2S)-2-((4-((4-((1R)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N,2-dimethyl-N′-(tetrahydro-2H-pyran-2-yloxy)malonamidewas obtained as a light brown foamy solid.

¹H-NMR (400 MHz, CDCl3)δ:1.53-1.69 (3H, m), 1.76-1.92 (31-1, m), 11.811,1.82 (3H, s), 2.27-2.37 (1H, m), 2.83-2.91 (4H, m), [3.17], 3.19 (3H,s), 3.53-3.83 (3H, m), [3.83-3.92], 3.98-4.08 (1H, m), 4.81-4.88 (1H,m), 4.94-5.04 (1H, m), 7.35 (2H, d, J=8.1 Hz), 7.45-7.59 (6H, m),[6.96-7.06], 7.59-7.68 (1H, m), [10.14], 10.56 (1H, s)

Production Example 8

To a mixture of 797 mg of(2S)-2-((4-((4-((1R)-1,2-dihydroxyethyl)phenyl)ethyny)benzoyl)(methyl)amino)-N,2-dimethyl-N-(tetrahydro-2H-pyran-2-yloxy)malonamide and 6.3 mL of methanol, 46 mg of p-toluenesulfonicacid monohydrate was added under ice cooling, and the resulting mixturewas stirred at the same temperature for 30 minutes and then at roomtemperature for 45 minutes. Water and ethyl acetate were added to thereaction mixture, and the resulting mixture was neutralized with asaturated aqueous solution of sodium hydrogen carbonate. The organiclayer was separated, and the aqueous layer was extracted with ethylacetate twice. Sodium chloride was added to the aqueous layer, and thesolid material was collected by filtration. Sodium chloride and ethylacetate were added to the filtrate, and the solid material was collectedby filtration. The organic layer of the filtrate was separated, theorganic layer, the extract, and the solid material thus obtained werecombined together, and then the solvent was distilled off under reducedpressure. The obtained residue was purified by silica gel columnchromatography [eluent; methanol:chloroform=10:90→15:85] to obtain 556mg of a yellow foamy solid. Ethyl acetate and IPE were added thereto,and the solid material was collected by filtration to obtain 458 mg of(2S)-2-((4-((4-((1R)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino-N-hydroxy-N′,2 -dimethyl-malonamide (Compound B) as a yellow solid.

¹H-NMR (400 MHz, CD3OD)δ: 1.78 (3H, s), 2.80 (3H, s), 3.17 (3H, s),3.57-3.67 (2H, m), 4.68-4.74 (1H, m), 7.42 (2H, d, J=8.3 Hz), 7.52 (2H,d, J=8.3 Hz), 7.56 (2H, d, J=8.6 Hz), 7.61 (2H, d, J=8.6 Hz); MS (ESI):462[M+Na]^(+,) 438[M−H]⁺

Production Example 9

To a mixture of 2.79 g of(1S)-1-(4-((triisopropylsilyl)ethynyl)ethane-1,2-diol, 28 mL ofdichloromethane, 2.7 mL of triethylamine, and 213 mg ofN,N-dimethylaminopyridine obtained in the same manner as in ProductionExample 3, 1.45 g of tert-butyldimethylsilyl chloride was added under anitrogen atmosphere and under ice cooling, and the resulting mixture wasstirred at room temperature for 2 hours, and then was allowed to standat the same temperature overnight. A saturated aqueous solution ofammonium chloride and ethyl acetate were added to the reaction mixture,and the pH was adjusted to 4.0 with 6 mol/L hydrochloric acid. Theorganic layer was separated, washed with a saturated aqueous solution ofsodium chloride, and then dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure to obtain 3.70 g of abrown oil.

To 3.70 g of the obtained brown oil, 28 ml, of dichloromethane and 439mg of pyridinium p-toluenesulfonate were added, 2.4 mL of3,4-dihydro-2H-pyran was added under ice cooling, and then the resultingmixture was stirred at room temperature for 5 hours. To the reactionmixture, 3.0 mL of triethylamine was added, and the solvent wasdistilled off under reduced pressure. Water and ethyl acetate were addedto the obtained residue. The organic layer was separated, washed with asaturated aqueous solution of sodium chloride, and then dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the obtained residue was purified by silica gel columnchromatography [eluent; diethyl ether:hexane=10:90] to obtain 3.65 g ofa yellow oil.

To 3.65 g of the obtained yellow oil, 18 mL of tetrahydrofuran wasadded, then 17 mL of a 1 mol/L solution of tetra-n-butylammoniumfluoride in tetrahydrofuran was added under ice cooling, and theresulting mixture was stirred at room temperature for 1 hour. Asaturated aqueous solution of ammonium chloride and ethyl acetate wereadded to the reaction mixture. The organic layer was separated, washedwith a saturated aqueous solution of sodium chloride, and then driedover anhydrous sodium sulfate. The solvent was distilled off underreduced pressure, and the obtained residue was purified by silica gelcolumn chromatography [eluent; ethyl acetate:hexane=30:70→40:60] toobtain 1.78 g of(2S)-2-(4-ethynylphenyl)-2-(tetrahydro-2H-pyran-2-yloxy)ethanol as awhite solid.

1H-NMR (400 MHz, CDCl3)δ:1.40-1.93 (614, m), 2.11-2.20 (1H, m), [3.06],3.07 (1H, s), 3.51-3.61 (1H, m), 3.62-3,76 (2H, m), [3.25-3.34],3.92-4.07 (1H, m), [4.48-4.53], 4.79-4.86 (1H, m), [4.70-4.75],4.87-4.93 (1H, m), [7.29], 7.35 (2H, d, J=8.3 Hz), 7.45 (2H, d, J=8.0Hz)

Production Example 10

To a mixture of 800 mg of(2S)-2-(4-ethynylphenyl)-2-(tetrahydro-2H-pyran-2-yloxy)ethanol, 4.0 mLof dimethyl sulfoxide, and 0.4 mL of methyl iodide, 545 mg of potassiumhydroxide was added under a nitrogen atmosphere and under ice cooling,and the resulting mixture was stirred at room temperature for 1 hour and30 minutes. Toluene and a saturated aqueous solution of ammoniumchloride were added to the reaction mixture, and the pH was adjusted to6.1 with 6 mol/L hydrochloric acid. The organic layer was separated,washed with a saturated aqueous solution of sodium chloride, and thendried over anhydrous magnesium sulfate. The solvent was distilled offunder reduced pressure, and the obtained residue was purified by silicagel column chromatography [eluent; ethyl acetate:hexane 10:90] to obtain836 mg of2-((1S)-1-(4-ethynylphenyl)-2-methoxyethoxy)tetrahydro-2H-pyran as acolorless oil.

1H-NMR (400 MHz, CDCl3)δ: 1.40-1.94 (6H, m), [3.05], 3.07 (1H, s),[3.36], 3.39 (3H, s), 3.45-3.56 (2H, m), [3.56-3.62], 3.62-3.69 (1H, m),[3.28-3.35], 3.97-4.06 (1H, m), [4.80-4.85], 4.91-4.97 (11-1, m),14.41-4.461, 4.97-5.01 (1H, m), [7.30], 7.37 (2H, d, J=8.4 Hz),7.44-7.51 (2H, m)

Production Example 11

To a mixture of 478 mg of2-((1S)-1-(4-ethynylphenyl)-2-methoxyethoxy)tetrahydro-2H-pyran, 3.0 mLof tetrahydrofuran, 300 mg of (2S)-2-((4-iodobenzoyl)(methy)amino)-N,2-dimethyl-N′-(tetrahydro-2H-pyran-2-yloxy)malonamide,43 mg of bis-triphenylphosphinepalladium(II) dichloride, and 23 mg ofcopper(I) iodide, 0.51 mL of triethylamine was added under a nitrogenatmosphere and under ice cooling, and the resulting mixture was stirredat the same temperature for 2 hours and 30 minutes. A saturated aqueoussolution of ammonium chloride and ethyl acetate were added to thereaction mixture, and the pH was adjusted to 6.0 with 6 mol/Lhydrochloric acid. The organic layer was separated, washed with asaturated aqueous solution of sodium chloride, and then dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the obtained residue was purified by silica gel columnchromatography [eluent; acetone:chloroform=10:90] to obtain 485 mg of(2S)-2-((4-((4-((1S)-2-methoxy-1-(tetrahydro-2H-pyran-2-yloxy)ethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N,2-dimethyl-N′-(tetrahydro-2H-pyran-2-yloxy)malonamide as a brown foamy solid.

¹H-NMR (400 MHz, CDCl3)δ: 1.42-1.94 (12H, m), [1.81], 1.82 (3H, s),[2.85], 2.86 (3H, d, J=4.4 Hz), [3.17], 3.20 (3H, s), [3.37], 3.40 (3H,s), 3.47-3.72 (4H, m), [3.29-3.36], 3.83-3.91 (1H, m), 3.97-4.07 (1H,m), [4.43-4.48], 4.93-4.98 (1H, m), [4.84], 4.95 (1H -1, dd, J=7.3, 4.2Hz), 4.98-5.03 (1H, m), [7.34], 7.41 (2H, d, J=8.3 Hz), 7.44-7.61 (6H,m), [6.96-7.04], 7.62-7.72 (1H, m), [10.01], 10.53 (1H, s)

Production Example 12

To a mixture of 485 mg of(2S)-2-((4-((4-((1S)-2-methoxy-1-(tetrahydro-2H-pyran-2-yloxy)ethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-2-dimethyl-N′-(tetrahydro-2H-pyran-2-yloxy)malonamideand 4.8 mL of methanol, 23 mg of p-toluenesulfonic acid monohydrate wasadded under ice cooling, and the resulting mixture was stirred at thesame temperature for 10 minutes and then at room temperature for 1 hour,Water and ethyl acetate were added to the reaction mixture, and theresulting mixture was neutralized with a saturated aqueous solution ofsodium hydrogen carbonate. The organic layer was separated, and theobtained aqueous layer was extracted with ethyl acetate. Sodium chloridewas added to the aqueous layer, and the aqueous layer was extracted withethyl acetate twice. The organic layer was combined with the extract,and then dried over anhydrous sodium sulfate. The solvent was distilledoff under reduced pressure, and the obtained residue was purified bysilica gel column chromatography [eluent; methanol:chloroform=4:96÷6:94]to obtain 288 mg of a brown solid. Ethyl acetate and IPE were addedthereto, and the solid material was collected by filtration to obtain240 mg of(2S)-N-hydroxy-2-((4-((4-((1S)-1-hydroxy-2-methoxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N′,2-dimethyl-malonamide (Compound C) as a brown solid.

1H-NMR (400 MHz, CD3OD)δ: 1.77 (3H, s), 2.79 (3H, s), 3.17 (3H, s), 3.37(3H, s), 3.50 (2H, d, J=5.9 Hz), 7.41 (2H, d, J=8.3 Hz), 7.47-7.65 (6H,m); MS (ESI): 476[M+Na]⁺, 452[M−H]⁻

Example 1

To a solution of 72.0 g of HPβCD (HPB-EC, NIHON SHOKUHIN KAKO CO., LTD.)in 130 mL, of water for injection, 7.2 g of Compound A monohydrate wasadded, and then stirred at room temperature to obtain an aqueoussolution of Compound A. To this solution, a portion of water forinjection was added to achieve a total amount of 200 mL. The resultantwas filtered through a 0.22 μm membrane filter to obtain a liquidformulation. The liquid formulation had a pH of 4.5.

Example 2

To 10 mL of the liquid formulation obtained in Example 1, 50 μL of 0.1mol/L hydrochloric acid and 250 μL of 0.01 mol/L hydrochloric acid wereadded. The resultant was filtered through a 0.22 μm membrane filter toobtain a liquid formulation. The liquid formulation had a pH of 3.0.

Example 3

To 10 mL of the liquid formulation obtained in Example 1, 70 μL of 0.01mol/L hydrochloric acid was added. The resultant was filtered through a0.22 μm membrane filter to obtain a liquid formulation. The liquidformulation had a pH of 4.0.

Example 4

To 10 mL of the liquid formulation obtained in Example 1, 20 μL of a0.01 mol/L sodium hydroxide aqueous solution was added. The resultantwas filtered through a 0.22 μm membrane filter to obtain a liquidformulation. The liquid formulation had a pH of 5.1.

Example 5

To 10 mL of the liquid formulation obtained in Example 1, 160 μL of a0.01 mol/L sodium hydroxide aqueous solution was added. The resultantwas filtered through a 0.22 μm membrane filter to obtain a liquidformulation. The liquid formulation had a pH of 6.0.

Example 6

To 10 mL of the liquid formulation obtained in Example 1, 90 μl, of a0.1 mol/L sodium hydroxide aqueous solution and 50 μL of a 0.01 mol/Lsodium hydroxide aqueous solution were added. The resultant was filteredthrough a 0.22 μm membrane filter to obtain a liquid formulation. Theliquid formulation had a pH of 7.0.

Example 7

To 10 mL, of the liquid formulation obtained in Example 1, 60 μL of a 1mol/L sodium hydroxide aqueous solution and 80 μL of a 0.1 mol/L sodiumhydroxide aqueous solution were added. The resultant was filteredthrough a 0.22 μm membrane filter to obtain a liquid formulation. Theliquid formulation had a pH of 8.0.

Example 8

To 10 mL of the liquid formulation obtained in Example 1, 40 μL of a 10%citric acid monohydrate aqueous solution was added. The resultant wasfiltered through a 0.22 μm membrane filter to obtain a liquidformulation. The liquid formulation had a pH of 3.0.

Example 9

To 10 mL of the liquid formulation obtained in Example 1, 2μL of a 10%citric acid monohydrate aqueous solution was added. The resultant wasfiltered through a 0.22 μm membrane filter to obtain a liquidformulation. The liquid formulation had a pH of 3.9.

Example 10

To 10 mL of the liquid formulation obtained in Example 1, 5 μL of a 1%meglumine aqueous solution was added. The resultant was filtered througha 0.22 μm membrane filter to obtain a liquid formulation. The liquidformulation had a pH of 5.1.

Example 11

To 10 mL of the liquid formulation obtained in Example 1, 25 μL, of a 1%meglumine aqueous solution was added. The resultant was filtered througha 0.22 μm membrane filter to obtain a liquid formulation. The liquidformulation had a pH of 5.9.

Example 12

To 10 mL of the liquid formulation obtained in Example 1, 20 μL of a 10%meglumine aqueous solution was added. The resultant was filtered througha 0.22 μm membrane filter to obtain a liquid formulation. The liquidformulation had a pH of 6.9.

Example 13

To 10 mL of the liquid formulation obtained in Example 1, 19 mg ofmeglumine was added and stirred. The resultant was then filtered througha 0.22 μm membrane filter to obtain a liquid formulation. The liquidformulation had a pH of 8.0.

Example 14

To a solution of 14.4 g HPβCD (HPB-EC, NIHON SHOKUHIN KAKO CO., LTD.) in25 mL of water for injection, 1.44 g of Compound A monohydrate wasadded, and then stirred at room temperature to obtain an aqueoussolution of Compound A. To this solution, a portion of water forinjection was added to achieve a total amount of 40 mL. The resultantwas filtered through a 0.22 μm membrane filter to obtain a preparation.To 5 mL of this preparation, a portion of 5% glucose aqueous solution(Otsuka Glucose Injection , Otsuka Pharmaceutical Factory, Inc.) wasadded to achieve a total amount of 100 mL. This preparation was frozenat −78° C. to obtain a frozen liquid formulation.

Example 15

To 10 mL of the preparation obtained in Example 14, a 5% glucose aqueoussolution (Otsuka Glucose Injection 5%, Otsuka Pharmaceutical Factory,Inc.) was added to achieve a total amount of 100 mL. This preparationwas frozen at −78° C. to obtain a frozen liquid formulation.

Example 16

To 20 mL of the preparation obtained in Example 14, a portion of 5%glucose aqueous solution (Otsuka Glucose Injection 5%, OtsukaPharmaceutical Factory, Inc.) was added to achieve a total amount of 100mL. This preparation was frozen at −78° C. to obtain a frozen liquidformulation.

Example 17

To a solution of 115.0 g of HPβCD (HPB-EC, NIHON SHOKUHIN KAKO CO.,LTD.) in 200 mL of water for injection, 11.5 g of Compound A monohydratewas added and then, stirred at room temperature to obtain an aqueoussolution of Compound A. To this solution, a portion of water forinjection was added to achieve a total amount of 320 mL. The resultantwas filtered through a 0.22 μm membrane filter to obtain a liquidpreparation. Two mL of this preparation was filled into a vial, andlyophilized. Then, the vial was hermetically sealed to obtain alyophilized formulation.

Example 18

To 23 mL of the liquid preparation obtained in Example 17, 2.31 g ofD-mannitol was added, and stirred. Then, the resultant was filteredthrough a 0.22 μm membrane filter to obtain a liquid preparation. Two mLof this preparation was filled into a vial, and lyophilized. Then, thevial was hermetically sealed to obtain a lyophilized formulation.

Example 19

To 23 mL of the liquid preparation obtained in Example 17, 2.31 g ofD-sorbitol was added, and stirred. Then, the resultant was filteredthrough a 0.22 μm membrane filter to obtain a liquid preparation. Two mLof this preparation was filled into a vial, and lyophilized. Then, thevial was hermetically sealed to obtain a lyophilized formulation.

Example 20

To 23 mL of the liquid preparation obtained in Example 17, 2.31 g ofxylitol was added, and stirred. Then, the resultant was filtered througha 0.22 μm membrane filter to obtain a liquid preparation. Two mL of thispreparation was filled into a vial, and lyophilized. Then, the vial washermetically sealed to obtain a lyophilized formulation.

Example 21

To 23 mL of the preparation obtained in Example 17, 2.30 g of trehalosewas added, and stirred. Then, the resultant was filtered through a 0.22μm membrane filter to obtain a liquid preparation. Two mL of thispreparation was filled into a vial, and lyophilized. Then, the vial washermetically sealed to obtain a lyophilized formulation.

Example 22

To 23 mL of the liquid preparation obtained in Example 17, 2.29 g ofglucose was added, and stirred. Then, the resultant was filtered througha 0.22 μm membrane filter to obtain a liquid preparation. Two mL of thispreparation was filled into a vial, and lyophilized. Then, the vial washermetically sealed to obtain a lyophilized formulation.

Example 23

To 23 mL of the liquid preparation obtained in Example 17, 2.31 g offructose was added, and stirred. Then, the resultant was filteredthrough a 0.2 μm membrane filter to obtain a liquid preparation. Two mLof this preparation was filled into a vial, and lyophilized. Then, thevial was hermetically sealed to obtain a lyophilized formulation.

Example 24

To 23 mL of the liquid preparation obtained in Example 17, 2.30 g ofsucrose was added, and stirred. Then, the resultant was filtered througha 0.22 μm membrane filter to obtain a liquid preparation. Two mL of thispreparation was filled into a vial, and lyophilized. Then, the vial washermetically sealed to obtain a lyophilized formulation.

Example 25

To 23 mL of the liquid preparation obtained in Example 17, 2.29 g ofglycine was added, and stirred. Then, the resultant was filtered througha 0.22 μm membrane filter to obtain a liquid preparation. Two mL of thispreparation was filled into a vial, and lyophilized. Then, the vial washermetically sealed to obtain a lyophilized formulation.

Example 26

To 24 mL of the liquid preparation obtained in the same manner as inExample 17, 40 μL of a 0.01 mol/L sodium hydroxide aqueous solution wasadded. This aqueous solution had a pH of 5.0. This aqueous solution wasfiltered through a 0.22 μm membrane filter to obtain a liquidpreparation. Two mL of this preparation was filled into a vial, andlyophilized. Then, the vial was hermetically sealed to obtain alyophilized formulation.

Example ¢

To 24 mL of the liquid preparation obtained in the same manner as inExample 17, 100 μL of a 0.1 mol/L sodium hydroxide aqueous solution, 40μL of a 0.01 mol/L sodium hydroxide aqueous solution and 95 μL of 0.1mol/L hydrochloric acid were added. This aqueous solution had a pH of5.0. This aqueous solution was filtered through a 0.22 μm membranefilter to obtain a liquid preparation. Two mL of this preparation wasfilled into a vial, and lyophilized. Then, the vial was hermeticallysealed to obtain a lyophilized formulation.

Example 28

To 24 mL of the liquid preparation obtained in the same manner as inExample 17, 10 μL of a 1% meglumine aqueous solution was added. Thisaqueous solution had a pH of 5.0. This aqueous solution was filteredthrough a 0.22 μm membrane filter to obtain a liquid preparation. Two mLof this preparation was filled into a vial, and lyophilized. Then, thevial was hermetically sealed to obtain a lyophilized formulation.

Example 29

To 24 mL of the liquid preparation obtained in the same manner as inExample 17, 20 μL of a 10% meglumine aqueous solution, 5 μL of a 1%meglumine aqueous solution and 10 μL of 0.1 mol/L hydrochloric acid wereadded. This aqueous solution had a pH of 5.0. This aqueous solution wasfiltered through a 0.22 μm membrane filter to obtain a liquidpreparation. Two mL of this preparation was filled into a vial, andlyophilized. Then, the vial was hermetically sealed to obtain alyophilized formulation.

Example 30

To 10 mL of the liquid formulation obtained in Example 1, 50 μL of 1mol/L hydrochloric acid and 350 μL of 0.1 mol/L hydrochloric acid wereadded to obtain a liquid formulation. The liquid formulation had a pH of2.0.

Example 31

To 10 mL of the liquid formulation obtained in Example 1, 300 μL of a 1mol/L sodium hydroxide aqueous solution was added to obtain a liquidformulation. The liquid formulation had a pH of 9.0.

Example 32

To 10 mL of the liquid formulation obtained in Example 1, 20 mg ofcitric acid monohydrate and 225 μL of a 10% citric acid monohydrateaqueous solution were added to obtain a liquid formulation. The liquidformulation had a pH of 2.0.

Example 33

To 10 mL of the liquid formulation obtained in Example 1, 100 mg ofmeglumine was added to obtain a liquid formulation. The liquidformulation had a pH of 9.0.

Examples 34 to 75

In accordance with the following experimental procedures, liquidformulations of Examples 34 to 75 were obtained.

Each abbreviation in tables has the following meaning.

Ac-L-Trp-OH: N-acetyl-L-tryptophan Ala: Alanine Arg: Arginine

BnOH: Benzyl alcohol

DMAc: N,N-dimethylacetamide DMSO: Dimethylsulfoxide EtOH: Ethanol His:Histidine

L-Phe-OBu(t)HCl: L-phenylalanine tert-butyl ester hydrochloridePEG: Polyethylene glycol

Phe: Phenylalanine Pro: Proline Trp: Tryptophan

β-Ala-OBu(t)HCl: β-alanine tert-butyl ester hydrochlorideExperimental procedure A

Compound A monohydrate, saline or water for injection, and asolubilizing agent, were mixed and then, stirred at room temperature toobtain a liquid formulation. When this liquid formulation was allowed tostand at room temperature for 2 hours, no precipitate was observed.

Experimental procedure B

Compound A monohydrate, saline or water for injection, and asolubilizing agent, were mixed and ultrasonicated to be dispersed. Thismixture was warmed to 40 to 50° C., to dissolve Compound A monohydrate,and then, left to room temperature to obtain a liquid formulation. Whenthis liquid formulation was allowed to stand at room temperature for 2hours, no precipitate was observed.

TABLE 1 Water for injection, Compound A monohydrate Example 34 35 36 3738 39 40 Experimental procedure A A A A B B B Compound A (mg) 20 20 4040 61 63 61 monohydrate Saline (mL) 1 1 1 1 Water for injection (mL) 1.21.2 1.2 BnOH (mL) 0.3 0.2 EtOH (mL) 0.5 0.3 DMAc (mL) 0.5 0.6 0.3 0.6PEG400 (mL) 1.2 1.2 Triethylene glycol (mL) 1.2 L-Arg (mg) 140Trometamol (mg) 600

TABLE 2 Water for injection, Compound A monohydrate Example 41 42 43 4445 46 Experimental procedure B A A A A A Compound A (mg) 61 40 40 40 4040 monohydrate Saline (mL) 1 1 1 1 1 Water for injection (mL) 1.2 BnOH(mL) 0.4 0.02 EtOH (mL) 0.3 DMAc (mL) 0.3 0.65 0.68 0.25 PEG400 (mL) 0.6Triethylene glycol (mL) 1.2 DMSO (mL) 0.6 Meglumine (mg) 17.2 17.3 NaOH(mg) 3.6 3.6

TABLE 3 Water for injection, Compound A monohydrate Example 47 48 49 5051 52 53 54 Experi- mental procedure A A A A A A A A Compound (mg) 40 4040 40 40 40 40 40 A mono- hydrate Saline (mL) 1 1 1 1 1 1 1 1 Water for(mL) injection EtOH (mL) DMAc (mL) 0.9 0.5 0.6 0.95 0.6 0.9 0.9 0.9PEG400 (mL) Meglumine (mg) 17.2 34.6 17.2 17.2 17.2 Benzoic (mg) 10.72.4 acid Sodium (mg) 12.7 12.6 benzoate Citric acid (mg) 18.7 Sodium(mg) 25.8 citrate Nicotinic (mg) 10.8 10.7 acid amide

TABLE 4 Water for injection, Compound A monohydrate Example 55 56 57 5859 60 Experimental procedure A B B B B B Compound A (mg) 40 20 20 21 2021 monohydrate Saline (mL) 1 0.7 Water for injection (mL) 0.5 0.4 0.70.6 EtOH (mL) 0.2 DMAc (mL) 0.9 0.3 0.1 0.3 0.4 PEG400 (mL) 0.4 0.4Benzoic acid (mg) 70 Sodium lactate (mg) 26.3 Nicotinic acid amide (mg)203 206 206 121

TABLE 5 Compound A monohydrate Example 61 62 63 64 65 66 67 68 Experi-mental procedure A A A A A A A A Compound (mg) 40 40 40 40 40 40 40 40 Amono- hydrate Saline (mL) 1 1 1 1 1 1 1 1 DMAc (mL) 0.9 0.9 0.9 0.9 0.90.9 0.9 0.9 L-His (mg) 13.7 L-Phe (mg) 14.7 L-Trp (mg) 18.0 DL-Pro (mg)10.2 β-Ala (mg) 7.9 D-Phe (mg) 14.5 D-Trp (mg) 17.9 Cinnamic (mg) 13.1acid

TABLE 6 Compound A monohydrate Example 69 70 71 72 73 74 75 Experimentalprocedure A A A A A A A Compound A (mg) 40 40 40 40 40 40 40 monohydrateSaline (mL) 1 1 1 1 1 1 1 DMAc (mL) 0.9 0.9 0.9 0.9 0.9 0.9 0.9 3- (mg)13.2 Phenylpropionic acid 3-(4- (mg) 14.7 Hydroxyphenyl) propionic acidAc-L-Trp-OH (mg) 21.6 β-Ala- (mg) 15.9 OBu(t)HCl L-Phe- (mg) 22.6OBu(t)HCl Taurine (mg) 11.0 p-Toluenesulfonic (mg) 16.9 acid monohydrate

Test Example 1 Test to Evaluate Pseudomonas aeruginosa LpxC EnzymeInhibitory Activity

Compound A, Compound B and Compound C were used as a test compound.

The Pseudomonas aeruginosa LpxC enzyme activity was measured by reactingLpxC with its substrateUDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine and measuring theamount of the reaction product by the quantification of an amino grouppresent in the product. This measurement was carried out according to amethod described in, for example, International Publication No. WO11/132712 pamphlet or a method similar thereto.

Specifically, to the Pseudomonas aeruginosa LpxC enzyme (which wasobtained by preparing chromosomal DNA from Pseudomonas aeruginosa,obtaining the Pseudomonas aeruginoss LpxC gene by PCR (polymerase chainreaction) using LpxC-specific primers, and incorporating this gene intoa vector, followed by gene expression using Escherichia coli). 20 μmol/LUDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine (Wako Pure ChemicalIndustries, Ltd.) was added, and the mixture was incubated at 25° C. for1 hour. This reaction was carried out in a 40 mmol/L HEPES buffersolution (pH 8.0) containing 20% Brij 35 and 80 μmol/L dithiothreitol.The reaction was terminated by the addition of 20% acetic acid (finalconcentration: 0.95%) to the reaction solution. Then, fluorescamine(final concentration: 1.6 mg/mL) dissolved in anhydrous dioxane wasadded thereto. The amount of the reaction product was detected at anexcitation wavelength/fluorescence wavelength=390 nm/495 nm. Each testcompound was allowed to coexist at various concentrations in thereaction to obtain an inhibition curve. From the inhibition curve, theconcentration at which the test compound inhibited 50% of the amount ofthe reaction product (IC₅₀ value) was determined and used as an indexfor Pseudomonas aeruginosa LpxC enzyme inhibitory activity.

As a result, all the IC₅₀ values of the test compounds were less than 50nM.

The test compounds exhibited an excellent Pseudomonas aeruginosa LpxCenzyme inhibitory activity.

Test Example 2 Test to Evaluate Antibacterial Activity

Compound A, Compound B and Compound C were used as a test compound.

The minimum inhibitory concentration (MIC) was measured according to theCLSI (Clinical and Laboratory Standards Institute) standard method usinga broth microdilution method given below.

The bacteria used were a Pseudomonas aeruginosa ATCC27853 strain.

Test bacterial cells of each strain cultured overnight in aMueller-Hinton agar medium were scraped off and suspended at theMcFarland 0.5 standard, and this suspension was diluted 10-fold toprepare an inoculum solution. The inoculum solution (0.005 mL) wasinoculated to a cation-adjusted Mueller-Hinton medium containing eachtest compound and cultured at 35° C. for 16 to 20 hours. The minimumdrug concentration at whith bacterial growth was not visible to thenaked eye was defined as MIC.

As a result, all the MICs of the test compounds were 1 μg/mL.

The test compounds exhibited an excellent antimicrobial activity againstPseudomonas aeruginosa.

Test Example 3 Test on Defense Against Mouse Systemic Infection usingPseudomonas aeruginosa

Compound A and Compound B were used as a test compound.

The mice used were male ICR SPF mice (4 weeks old: 5 individuals pergroup).

To prepare a bacterial inoculum solution, a Pseudomonas aeruginosaclinical isolate (S-3232 strain) cultured overnight at 37° C. on aMueller-Hinton agar plate was cultured for 4 hours in a cation-adjustedMueller-Hinton medium and then diluted 10-fold with a 10%mucin/phosphate buffer solution to prepare the inoculum solution.

Infection was induced by the intraperitoneal inoculation of 0.5 mL ofthe inoculum solution (approximately 10⁴ CFU/mouse) to each mouse. Eachtest compound was dissolved in a 10% HPβCD /2.5% mannitol aqueoussolution and subcutaneously administered a single dose of 12.5 mg/kg at1 hour after the infection. Three days after the infection, the numberof survivors was recorded.

As a result, in the control group wherein the test compound was notadministered, all the mice died. In the groups wherein the test compoundwas administered, 80% or more of the mice were observed to survive 3days after the bacterial inoculation, and an in vivo anti-Pseudomonasaeruginosa activity was confirmed. Also, in the group wherein 6.25 mg/kgof the test compound was administered, 80% or more of the mice wereobserved to survive 3 days after the bacterial inoculation, and anexcellent in vivo anti-Pseudomonas aeruginosa activity was confirmed.

Test Example 4 Test on Defense Against Mouse Systemic Infection usingMultidrug-Resistant Pseudomonas aeruginosa

Compound A, Compound B and Compound C were used as a test compound.

The mice used were male ICR SPF mice (4 weeks old: 5 individuals pergroup).

To prepare a bacterial inoculum solution, a multidrug-resistantPseudomonas aeruginosa clinical isolate (S-2838 strain) culturedovernight at 37° C. on a Mueller-Hinton agar plate was cultured for 5hours in a cation-adjusted Mueller-Hinton medium and then diluted10-fold with a 10% mucin/phosphate buffer solution to prepare theinoculum solution.

Infection was induced by the intraperitoneal inoculation of 0.5 mL ofthe inoculum solution (approximately 10⁶ CFU/mouse) to each mouse. Eachtest compound was dissolved in a 10% HPβCD /2.5% mannitol aqueoussolution and intravenously administered to the tail a single dose of 50mg/kg at 1 hour after the infection. Three days after the infection, thenumber of survivors was recorded.

As a result, in the control group wherein the test compound was notadministered, all the mice died. In all the groups wherein the testcompound was administered, 100% of the mice were observed to survive 3days after the bacterial inoculation, and an in vivoanti-multidrug-resistant Pseudomonas aeruginosa activity was confirmed.Also, in the group wherein 25 mg/kg of Test Compound A was administered,60% or more of the mice were observed to survive 3 days after thebacterial inoculation, and an excellent in vivo anti-multidrug-resistantPseudomonas aeruginosa activity was confirmed.

Test Example 5 Test on Mouse Model with Urinary Tract Infection byMultidrug-Resistant Pseudomonas aeruginosa

Compound A, Compound B and Compound C were used as a test compound.

The mice used were female ICR SPF mice (5 weeks old: 5 individuals pergroup).

To prepare a bacterial inoculum solution, a Pseudomonas aeruginosaclinical isolate (S-2838 strain) was suspended in sterile saline.

Infection was induced by the inoculation of 0.2 mL, of the inoculumsolution (approximately 10³ CFU/mouse) through the urethra of eachmouse. Each test compound was dissolved in a 10% HPβCD/2.5% mannitolaqueous solution and intravenously administered to the tail at a dose of25 mg/kg once 2 hours after the infection. The numbers of bacterialcolonies of the next day of the infection in the kidneys were recorded,and an average thereof was calculated.

As a result, in all the groups wherein the test compound wasadministered, as compared to the control group wherein the test compoundwas not administered, a decrease of 2 log CFU/kidney or more in theintrarenal viable cell count was observed, and an anti-Psendomonasaeruginosa activity in the urinary tract infection model was confirmed.Also, in the group wherein 12.5 mg/kg of Test Compound A wasadministered, as compared to the control group wherein the test compoundwas not administered, a decrease of 2 log CFU/kidney or more in theintrarenal viable cell count was observed, and an excellentanti-Pseudomonas aeruginosa activity in the urinary tract infectionmodel was confirmed.

Test Example 6 Test on Mouse Model with Pulmonary Infection byMultidrug-Resistant Psendomonas aeruginosa

Compound A, Compound B and Compound C were used as a test compound.

The mice used were male ICR SPF mice (4.5 weeks old at the time ofinfection: 5 individuals per group). In order to achieve a transientcompromised state, cyclophosphamide was intraperitoneally administeredat a dose of 200 mg/kg to each mouse 4 days before injection.

To prepare a bacterial inoculum solution, a Pseudomonas aeruginosaclinical isolate (S-2838 strain) was suspended in sterile saline.

Infection was induced by the inoculation of 0.05 mL of the inoculumsolution (approximately 10⁵ CFU/mouse) to each mouse intranasally. Eachtest compound was dissolved in a 10% HPβCD /2.5% mannitol aqueoussolution and intravenously administered to the tail at a dose of 50mg/kg twice 2 and 8 hours after the infection. The numbers of bacterialcolonies of the next day of the infection in the lungs were recorded,and an average thereof was calculated.

As a result, in all the groups wherein the test compound wasadministered, as compared to the control group wherein the test compoundwas not administered, a decrease of 2 log CFU/lung or more in theintrapulmonary viable cell count was observed, and an anti-Pseudomonasaeruginosa activity in the pulmonary infection model was confirmed.Also, in the group wherein 25 mg/kg of Test Compound A was administered,as compared to the control group wherein the test compound was notadministered, a decrease of 2 log CFU/lung or more in the intrapulmonaryviable cell count was observed, and an excellent anti-Pseudomonasaeruginosa activity in the pulmonary infection model was confirmed.

Test Example 7 Test on Inhibition of Vero Cell Growth

Compound A, Compound B and Compound C were used as a test compound.

Each test compound was dissolved in dimethyl sulfoxide, adjusted to eachconcentration using E'MEN, and then dispensed at 0.1 mL/well to 96-wellmicroplates. The Vero cell suspension was prepared at 3×10⁴ cells/mLusing E'MEM supplemented with 20% FBS, inoculated thereto at 0.1mL/well, and cultured at 37° C. for 3 days under 5% CO₂. At thecompletion of the culture, PBS supplemented with 1 mg/mL2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazoliuminner salt monosodium salt (XTT) and 25 μM phenazine methosulfate (PMS)was prepared and added thereto at 50 μL/well. Approximately 2 hourslater, the absorbance at 450 nm was measured using a microplate reader.

The absorbance ratio between a test compound-non-supplemented controland each well was calculated to calculate the concentration at which thecompound inhibited 50% of cell growth (CC₅₀; μg/mL).

As a result, all the CC50s of the test compounds were 100 μg/mL or more.

Test Example 8 Evaluation of hERG Inhibitory Activity

Compound A and Compound C were used as a test compound.

HEK 293 cells (human embryo kidney 293 cells, Cytomyx LLC) transfectedwith hERG gene (human ether-a-go-go related gene) were used.

The culture solution used was a MEM medium containing 10% fetal bovineserum and 1% non-essential amino acid and further supplemented withGeneticin at a concentration of 400 μg/mL. The cells were cultured in acarbonic acid gas incubator (37.0° C., 5% CO₂).

The hERG current was measured by a whole cell clamp method. A glasscover with the cells for measurement attached thereto was placed in adish and perfused at a rate of 2 mL/min with a perfusate (composition:137 mmol/L NaCl, 4 mmol/L KCl, 10 mmol/L HEPES, 1.8 mmol/L CaCl₂, 1mmol/L MgCl₂, 10 mmol/L glucose, pH 7.4). The inside temperature of theperfusion chamber was kept at 25° C. The cells were contacted with aglass electrode (2.0 to 8.0 MΩ) charged with an internal solution(composition: 130 mmol/KCl, 1 mmol/L MgCl₂, 5 mmol/L EGTA, 10 mmol/LHEPES, 5 mmol/L MgATP, pH 7.2) to break the patch membranes, followed bythe measurement of the hERG current using a patch clamp amplifier (EPC-7Plus, HEKA) via patch clamp software pClamp 10 (Molecular DevicesCorporation). The pulse protocol involved a holding potential of −80 mV,a depolarizing pulse of +20 mV for 1.5 seconds and a repolarizing pulseof −50 mV for 1.5 seconds. After confirmation that a stable currentwaveform was obtained, each test compound was applied thereto.

Before the application and 10 minutes after the application, the peakvalue of tail current in the hERG current waveform was analyzed tocalculate the ratio of the value 10 minutes after the application to thevalue before the application (relative value, %).

As a result, none of the test compounds exhibited an hERG inhibitoryactivity up to 300 μmol/L.

Test Example 9 In vitro Micronucleus Test for Examining the Presence orAbsence of Genotoxicity

Compound A was used as a test compound.

In order to examine the inducibility of the chromosomal aberrations byeach test compound in cultured cells, the in vitro micronucleus test wascarried out. This test was carried out by a short-time treatment method(in the presence and absence of a metabolic activation) and a 30-hourtreatment method using Chinese hamster lung fibroblasts (CHL/IU cells).The concentration of the test compound was set to 1.00 mmol/L as themaximum dose with reference to the “Guidance on Genotoxicity Testing andData Interpretation for Pharmaceuticals Intended for Human Use”.Specimens were observed as to doses of 0.25, 0.50 and 1.00 mmol/L.

The cells were inoculated at 15×10⁴ cells to a 60-mm dish (MAKI) andprecultured at 37° C. for 24 hours under 5% CO₂ using a MEM medium(Sigma-Aldrich Co., Ltd.) containing 10% newborn calf serum(Sigma-Aldrich Co., Ltd.) and 50 U/mL-50 μg/mL Penicillin-Streptomycin(Sigma-Aldrich Co., Ltd.). After the completion of the preculture avehicle (DMSO) or each test compound was added thereto. In theshort-time treatment method, 6 hours after the culture, the cells werewashed with PBS(-) (Sigma-Aldrich Co., Ltd,), and then, the medium wasreplaced with a fresh medium, followed by further culture for 24 hours.In the 30-hour treatment method, after the addition of the testcompound, the cells were cultured for 30 hours. After the completion ofthe culture, the cells were dissociated using a 0.05% trypsin-EDTAsolution (Sigma-Aldrich Co., Ltd.). After centrifugation, thesupernatant was removed, and 3 mL of a 0.075 mol/L aqueous potassiumchloride solution was added to the cells. After hypotonic treatment atroom temperature for 5 minutes, the cells were fixed with an ice-coldfixing solution (methanol:acetic acid=19:1) to prepare a glass slidespecimen (giemsa-stained (Merck)). Two thousand cells per dose wereobserved to measure the number of cells having the micronucleus. Whenthe frequency of appearance of the micronucleus in the test compoundgroup was significantly increased as compared with the vehicle controlgroup, the test compound was confirmed to be positive. When thisfrequency of appearance was equivalent to that of the vehicle control,the test compound was confirmed to be negative.

As a result, in either treatment method, the test compound was negativeat the dose of 1 mmol/L or less.

Test Example 10 Measurement of Binding Ratio to Plasma Protein

Compound A and Compound C were used as a test compound.

Each test compound was added to human serum to prepare a 1 μg/mL spikedserum, which was then left standing at room temperature for 1 hour orlonger. A filtrate (20 μL) was collected by a centrifugalultrafiltration method (molecular weight cutoff: 10,000, 1500×g, 25° C.,10 min), then human serum and an internal standard solution(furosemide-acetonitrile solution) were added thereto. To thecompound-spiked serum, PBS and an internal standard solution were added.Each mixture was stirred and then centrifuged, and the concentration inthe supernatant was determined by LC-MS/MS.

The ratio of protein binding was determined according to the followingcalculation expression:

Ratio of protein binding (%)=(1−(Concentration of thefiltrate)/(Concentration of the compound-spiked serum))×100

As a result, all the protein binding ratios of the test compounds were80% or less.

Test Example 11 Inhibitory Effect on Liver Drug-Metabolizing Enzyme inHuman

Compound A and Compound C were used as a test compound.

Pooled human liver microsomes were used. Substrates and their finalconcentrations as well as the positive controls and their finalconcentrations were as described in Tables 7 and 8. The reaction wascarried out in a phosphate buffer solution (100 mmol/L, pH 7.4), and thefinal concentrations of the reaction system were set to 0.5 mg/mL humanliver microsome protein, 1.55 mmol/L oxidized form of nicotinamideadenine dinucleotide phosphate (NADP+), 3.3 mmol/L glucose-C-phosphate.3.3 mmol/L magnesium chloride and 0.4 Units/mL glucose-6-phosphatedehydrogenase (G6PDH). The final concentration of each compound in thereaction solution was set to 100 μM. Each of these reaction solutionswas incubated at 37° C. for 30 minutes. Then, the substrates were addedthereto and reacted at 37° C. for 10 minutes. The reaction wasterminated by the addition of a 1.5-fold volume of an internal standardsolution acetonitrile solution containing 0.25 mmol/L dextrorphan and 2%formic acid). Then, the solution was centrifuged, and the concentrationof metabolites in the supernatant was determined by LC-MS/MS.

The ratio of inhibitory activity by addition of the inhibitor wasdetermined according to the following calculation expression:

Ratio of inhibitory activity (%)=(1−(Concentration of CYP metabolites inthe presence of the test compound)/(Concentration of CYP metabolites inthe absence of the test compound))×100

As a result, all the inhibitory activity ratios of the test compoundswere 30% or less.

TABLE 7 Final Molecular concentration species Substrate name (μmol/L)CYP1A2 Phenacetin 10 CYP2C8 Amodiaquine 0.2 CYP2C9 Tolbutamide 100CYP2C19 (S)-Mephenytoin 40 CYP2D6 (±)-Bufuralol 4 CYP3A4 Midazolam 1CYP3A4 Testosterone 5

TABLE 8 Final Molecular concentration species Positive control (μmol/L)CYP1A2 Furafyline 10 CYP2C8 Quercetin 10 CYP2C9 Tienilic acid 1 CYP2C19Ticlopidine 1 CYP2D6 Paroxetine 2 CYP3A4 Verapamil 10

Test Example 12 Stability Test

The liquid formulations obtained in Examples 2 to 13 and 30 to 33 werestored at 25° C. for 24 hours. The concentration of Compound A afterstorage was measured by the HPLC method to determine the residual ratio.

The results are shown in Table 9.

The residual ratio was determined by the following expression.

Residual ratio (%)=(Concentration of Compound A after storage /Concentration of Compound A at the beginning of the test)×100

<HPLC Measurement Conditions> Detector: LC-2010CHT (SHILMADZUCORPORATION) Detection at: 254 nm Column: XBridge C18 4.6×150 min(Waters Corporation) Precolumn: Develosil ODS-HG 4.0×10 mm (NomuraChemical Co., Ltd.)

Column temperature: 40° C.Flow rate: 1.0 mL/minuteMobile phase A: water/(0.2 mol/L formate buffer solution (pH 3))=90/10Mobile phase B: acetonitrile/(0.2 mol/L formate buffer solution (pH3))=90/10Gradient cycle: 0 min (A solution/B solution 90/10), 15 min (Asolution/B solution=70/30), 20 min (A solution/B solution =0/100), 30min (A solution/B solution =0/100)

TABLE 9 Residual pH ratio (%) Example 2 3.0 96.9 Example 3 4.0 99.4Example 4 5.1 100.0 Example 5 6.0 99.4 Example 6 7.0 98.8 Example 7 8.097.8 Example 8 3.0 97.6 Example 9 3.9 99.7 Example 10 5.1 101.9 Example11 5.9 100.4 Example 12 6.9 98.2 Example 13 8.0 101.0 Example 30 2.093.0 Example 31 9.0 92.6 Example 32 2.0 93.5 Example 33 9.0 92.1

The residual ratios of the liquid formulations in Examples were 90% ormore. Particularly, the residual ratios of the liquid formulations inExamples in which the pH was from 3 to 8 were 95% or more. The liquidformulations in Examples were stable.

Test Example 13 Solubility Test

Compound A monohydrate was used as a test compound.

To 5 mL of a 10% CD or CD derivative solution, about 100 mg of CompoundA monohydrate was added, and stirred at room temperature for 24 hours.To the solutions in which αCD, HPαCD, DMβCD and methyl-β-cyclodextrinwere used, about 100 mg of Compound A monohydrate was added after 6hours. After centrifugation (3000 rpm, 10 minutes), the supernatant wasfiltered with a filter, and the solubility was measured by the HPLCmethod.

The results are shown in Table 10.

TABLE 10 Solubility CD or CD derivative (mg/mL) None 0.2 αCD (NIHONSHOKUHIN 30.2 KAKO CO., LTD.) βCD (NIHON SHOKUHIN 5.9 KAKO CO., LTD.)γCD (ASHLAND) 14.4 HPαCD (NIHON SHOKUHIN 25.7 KAKO CO., LTD.) SBEβCD(ChemScene) 8.9 2,3,6-Tri-O-methyl-β- (Wako Pure Chemical 4.7cyclodextrin Industries, Ltd.) Hydroxyethyl-β- (Sigma-Aldrich) 11.9cyclodextrin HPβCD (NIHON SHOKUHIN 11.6 KAKO CO., LTD.) DMβCD(Sigma-Aldrich) 16.6 6-O-α-maltosyl-β- (Wako Pure Chemical 10.0cyclodextrin Industries, Ltd.) Methyl-β-cyclodextrin (Sigma-Aldrich)17.5 HPγCD (ASHLAND) 8.8

Compound A exhibited excellent solubility due to the CD or CDderivative.

Industrial Applicability

The pharmaceutical composition comprising the hydroxamic acid derivativeof the present invention or a salt thereof and a solubilizing agentexhibits a potent antimicrobial activity, has excellent solubility, andis useful as a medicine.

1. A pharmaceutical composition comprising (i) a hydroxamic acidderivative selected from the group consisting of:(2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-hydroxy-N′,2-dimethylmalonamide,(2S)-2-((4-((4-((1R)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-hydroxy-N′,2-dimethylmalonamideand2(S)-N-hydroxy-2-((4-((4-((1S)-1-hydroxy-2-methoxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N′,2-dimethylmalonamide,or a salt thereof, and (ii) a solubilizing agent, with the proviso thata 10% hydroxypropylated β-cyclodextrin/2.5% mannitol aqueous solution ofthe hydroxamic acid derivative or a solution of the hydroxamic acidderivative in dimethyl sulfoxide are not covered.
 2. The pharmaceuticalcomposition according to claim 1, wherein the solubilizing agent is acyclodextrin or a cyclodextrin derivative.
 3. The pharmaceuticalcomposition according to claim 1, wherein the hydroxamic acid derivativeis(2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino-N-hydroxy-N′,2-dimethylmalonamide.
 4. The pharmaceutical composition according to claim2, wherein the solubilizing agent is one or more selected from the groupconsisting of α-cyclodextrin, γ-cyclodextrin,hydroxypropyl-α-cyclodextrin, sulfobutylether-β-cyclodextrin,2,3,6-tri-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin,hydroxypropyl-β-cyclodextrin, heptakis-2,6-di-O-methyl-β-cyclodextrin,6-O-α-maltosyl-β-cyclodextrin, methyl-β-cyclodextrin andhydroxypropyl-γ-cyclodextrin.
 5. The pharmaceutical compositionaccording to claim 2, wherein the solubilizing agent is one or moreselected from the group consisting of α-cyclodextrin, γ-cyclodextrin,hydroxypropyl-α-cyclodextrin, sulfobutylether-β-cyclodextrin,hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin.
 6. Thepharmaceutical composition according to claim 1, wherein thepharmaceutical composition is a liquid formulation.
 7. Thepharmaceutical composition according to claim 6, wherein a pH of theliquid formulation is from 3 to
 8. 8. The pharmaceutical compositionaccording to claim 1, wherein the pharmaceutical composition is a frozenliquid formulation.
 9. The pharmaceutical composition according to claim8, wherein a pH of the frozen liquid formulation when thawed is from 3to
 8. 10. The pharmaceutical composition according to claim 1, whereinthe pharmaceutical composition is a lyophilized formulation.
 11. Thepharmaceutical composition according to claim 10, wherein a pH of anaqueous solution of the lyophilized formulation is from 3 to
 8. 12. Thepharmaceutical composition according to claim 1, wherein thesolubilizing agent is one or more selected from the group consisting ofmonoalcohols, polyhydric alcohols, amides, sulfoxides, amino acids,surfactants, acids and bases.
 13. The pharmaceutical compositionaccording to claim 12, wherein the hydroxamic acid derivative is(2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-hydroxy-N′,2-dimethylmalonanide.14. The pharmaceutical composition according to claim 12, wherein themonoalcohol is an alcohol having 1 to 6 carbon atoms; the polyhydricalcohol is a diol; and the acid is an organic acid.
 15. Thepharmaceutical composition according to claim 12, wherein thesolubilizing agent is one or more selected from the group consisting ofalcohols having 1 to 6 carbon atoms, diols, amino acids and organicacids in combination with an amide.
 16. The pharmaceutical compositionaccording to claim 12, wherein the pharmaceutical composition is aliquid formulation.
 17. A method of inhibiting LpxC, comprisingadministering the pharmaceutical composition according to claim 1 to asubject in need thereof.
 18. A method of inhibiting Gram-negativebacteria, comprising administering the pharmaceutical compositionaccording to claim 1 to a subject in need thereof.
 19. A method forproducing a liquid formulation comprising a hydroxamic acid derivativeor a salt thereof and a solubilizing agent, the method comprising:dissolving the hydroxamic acid derivative selected from the groupconsisting of(2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-hydroxy-N′,2-dimethylmalonamide,(2S)-2-((4-((4-((1R)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino-N-hydroxy-N′,2-dimethylmalonamideand(2S)-N-hydroxy-2-((4-((4-((1S)-1-hydroxy-2-methoxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N′,2-dimethylmalonamide,or the salt thereof, and the solubilizing agent in water to obtain anaqueous solution of the hydroxamic acid derivative or the salt thereof,with the proviso that a 10% hydroxypropylated β-cyclodextrin/2.5%mannitol aqueous solution of the hydroxamic acid derivative or asolution of the hydroxamic acid derivative in dimethyl sulfoxide are notcovered, followed by adjusting a pH of the obtained aqueous solution to3 to
 8. 20. The production method according to claim 19, wherein thesolubilizing agent is a cyclodextrin or a cyclodextrin derivative. 21.The production method according to claim 19, wherein the hydroxamic acidderivative is(2S)-2-((4-((4-((1S)-1,2-dihydroxyethyl)phenyl)ethynyl)benzoyl)(methyl)amino)-N-hydroxy-N′,2-dimethylmalonanmide.
 22. The production method according to claim 20,wherein the solubilizing agent is one or more selected from the groupconsisting of α-cyclodextrin, γ-cyclodextrin,hydroxypropyl-α-cyclodextrin, sulfobutylether-β-cyclodextrin,2,3,6-tri-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin,hydroxypropyl-β-cyclodextrin, heptakis-2,6-di-O-methyl-β-cyclodextrin,6-O-α-maltosyl-β-cyclodextrin, methyl-β-cyclodextrin andhydroxypropyl-γ-cyclodextrin.
 23. The production method according toclaim 20, wherein the solubilizing agent is one or more selected fromthe group consisting of α-cyclodextrin, γ-cyclodextrin,hydroxypropyl-α-cyclodextrin, sulfobutylether-β-cyclodextrin,hydroxypropyl-β-cycodextrin and hydroxypropyl-γ-cyclodextrin.